Hepatocyte lineage cells derived from pluripotent stem cells

ABSTRACT

It has been discovered that when pluripotent stem cells are cultured in the presence of a hepatocyte differentiation agent, a population of cells is derived that has a remarkably high proportion of cells with phenotypic characteristics of liver cells. In one example, human embryonic stem cells are allowed to form embryoid bodies, and then combined with the differentiation agent n-butyrate, optionally supplemented with maturation factors. In another example, n-butyrate is added to human embryonic stem cells in feeder-free culture. Either way, a remarkably uniform cell population is obtained, which is predominated by cells with morphological features of hepatocytes, expressing surface markers characteristic of hepatocytes, and having enzymatic and biosynthetic activity important for liver function. Since stem cells readily proliferate in culture, this system provides an abundant source of cells of the hepatocyte lineage for a variety of applications, such as drug screening, and replenishing liver function in the context of clinical treatment.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of International Patent Applicationwith the same title filed on Apr. 26, 2001 PCT/US01/13471, to bepublished in English 18 months after the first priority date pursuant toArticle 21(2) of the PCT. This application also claims priority to U.S.provisional patent application No. 60/200,095, filed Apr. 27, 2000 andis a continuation of application Ser. No. 09/718,308 filed Nov. 20,2000, now U.S. Pat. No. 6,458,589. The two priority applications andU.S. utility application Ser. No. 09/718,308 are hereby incorporatedherein by reference in their entirety.

TECHNICAL FIELD

This invention relates generally to the field of cell biology ofembryonic cells and liver cells. More specifically, this inventionrelates to the directed differentiation of human pluripotent stem cellsto cells of the hepatocyte lineage under special culture conditions.

BACKGROUND

Liver disease affects millions of people worldwide. Fulminant hepaticfailure is the clinical term for an immediate and catastrophic cessationin liver function, usually leading to death within a matter of hours.Other forms of liver disease, such as chronic hepatitis and cirrhosis,involve an insidious and progressive failure of liver function, withgrim effects on physiological well-being and long-term prognosis. In theUnited States, there are an estimated 300,000 hospitalizations each yearfor liver disease, and 30,000 deaths—with only about 4,500 donor liversavailable for transplant.

A healthy liver has a remarkable ability to regenerate itself—but whenthis ability is compromised, the consequences are dire. An importantchallenge of modern medicine is to find a way to supplement the naturalprocess of regeneration, and thereby restore liver function to affectedpatients.

Some early work has been done to identify liver progenitor cells insmall animal models. Agelli et al. (Histochem. J. 29:205, 1997), Brillet al. (Dig. Dis. Sci. 44:364, 1999 and), and Reid et al. (U.S. Pat. No.5,576,207) have proposed expansion conditions for early hepaticprogenitor cells from embryonal and neonatal rat livers. Michalopouloset al. (Hepatology 29:90, 1999) report a system for culturing rathepatocytes and nonparenchymal cells in biological matrices. Block etal. (J. Cell Biol. 132:1133, 1996) developed conditions for expansion,clonal growth, and specific differentiation in primary cultures ofhepatocytes induced by a combination of growth factors in a chemicallydefined medium. It has been known for some time that mature rat livercells derive from precursors (sometimes referred to as “hepatoblasts” or“oval cells”) that have the capacity to differentiate into either maturehepatocytes or biliary epithelial cells (L. E. Rogler, Am. J. Pathol.150:591, 1997; M. Alison, Current Opin. Cell Biol. 10:710, 1998; Lazaroet al., Cancer Res. 58:514,1998; Germain et al., Cancer Res. 48:4909,1988).

Unfortunately, a ready source of human hepatocytes for reconstitutiontherapy has not been identified. European Patent Application EP 953 633A1 proposes a cell culturing method and medium for producingproliferated and differentiated human liver cells, apparently fromdonated human liver tissue. In most people's hands, the replicationcapacity of human hepatocytes in culture has been disappointing. As aremedy, it has been proposed that hepatocytes be immortalized bytransfecting with large T antigen of the SV40 virus (U.S. Pat. No.5,869, 243).

A number of recent discoveries have raised expectations that stem cellsmay become a source of a variety of cell types and tissues for replacingthose damaged in the course of disease, infection, or from congenitalabnormalities. Various types of putative stem cells differentiate asthey divide, maturing into cells that can carry out the unique functionsof particular tissues, such as the heart, the liver, or the brain.

A particularly important development has been the isolation of two typesof human pluripotent stem (hPS) cells from embryonic tissue. Pluripotentcells are believed to have the capacity to differentiate into most celltypes in the body (R. A. Pedersen, Scientif. Am. 280(4):68, 1999). Earlywork on embryonic stem cells was done in mice (reviewed in Robertson,Meth. Cell Biol. 75:173, 1997; and Pedersen, Reprod. Fertil. Dev. 6:543,1994). However, monkey and human pluripotent cells have proven to bemuch more fragile, and do not respond to the same culture conditions asmouse embryonic cells. It is only recently that discoveries were madethat allow primate embryonic cells to be obtained and cultured ex vivo.

Thomson et al. (U.S. Pat. No. 5,843,780; Proc. Natl. Acad. Sci. USA92:7844, 1995) were the first to successfully culture embryonic stemcells from primates. They subsequently derived human embryonic stem(hES) cell lines from human blastocysts (Science 282:114, 1998).Gearhart and coworkers derived human embryonic germ (hEG) cell linesfrom fetal gonadal tissue (Shamblott et al., Proc. Natl. Acad. Sci. USA95:13726, 1998 and International Patent Application WO 98/43679). BothhES and hEG cells have the long-sought characteristics of humanpluripotent stem (hPS) cells: they are capable of ongoing proliferationin vitro without differentiating, they retain a normal karyotype, andthey retain the capacity to differentiate to produce all adult celltypes.

Spontaneous differentiation of pluripotent stem cells in culture or interatomas generates cell populations with a heterogeneous mixture ofphenotypes, representing a spectrum of different cell lineages. In anumber of applications, it is desirable for differentiated cells to beof a more homogeneous nature—both in terms of the phenotypes theyexpress, and in terms of the types of progeny they can generate.

Accordingly, there is a need for technology to generate more homogeneousdifferentiated cell populations from pluripotent embryonic cells ofprimate origin, particularly those from humans.

SUMMARY

This invention provides a system for efficient production of primatecells that have differentiated from pluripotent cells into cells of thehepatocyte lineage. Cultures of such cells have been obtained that arerelatively enriched for characteristics typical of liver cells, comparedwith undifferentiated cells and cells that are committed to other tissuetypes.

One embodiment of the invention is a cell population obtained bydifferentiating primate pluripotent stem (pPS) cells in such a mannerthat a significant proportion of cells in the population havecharacteristics of cells of the hepatocyte lineage. Desirablecharacteristics are listed later in the description. The cells maydemonstrate any or all of the following: antibody-detectable expressionof α₁-antitrypsin or albumin; absence of antibody-detectable expressionof α-fetoprotein; expression of asialoglycoprotein receptor at a leveldetectable by reverse PCR amplification; evidence of glycogen storage;evidence of cytochrome p450 or glucose-6-phosphatase activity; andmorphological features of hepatocytes. Preferred cell populations havemore of these hepatocyte characteristics in a greater proportion of thecells in the population. It is understood that the cells may replicateto form progeny, both during differentiation, and in subsequentmanipulation. Such progeny also fall within the scope of the inventionin all instances where not explicitly excluded.

Exemplary cells are obtained by differentiating embryonic stem (hES)cells obtained from cultures that originated from human blastocysts. Thedifferentiated cells are generated by culturing the pPS cells in agrowth environment that comprises a hepatocyte differentiation agent,such as n-butyric acid or other differentiation agent outlined in thedisclosure. The differentiation agent can be added directly toundifferentiated pPS cells cultured with or without feeder cells.Alternatively, the pPS cells are allowed to differentiate into a mixedcell population (e.g., by forming embryoid bodies or by cultureovergrowth), and the differentiation agent is added to the mixedpopulation. What emerges is a less heterogeneous population, in which asubstantial proportion of the cells have the desired phenotype. In someinstances, the culture method also includes hepatocyte maturationfactors such as those exemplified in the disclosure, which includesolvents like DMSO, growth factors like FGF, EGF, and hepatocyte growthfactor, and glucocorticoids like dexamethazone.

Another embodiment of the invention is a differentiated cell havingcharacteristics of a cell of the hepatocyte lineage, which is eitherharvested from a differentiated cell population of this invention, or isthe progeny of a cell harvested from such a population. Exemplary is adifferentiated cell produced by providing a human pluripotent stem (hPS)cells in a growth environment essentially free of feeder cells;culturing the hPS cells in a medium containing a hepatocytedifferentiation agent under conditions that produce a cell populationenriched for cells with characteristic features of hepatocytes; andsubsequently harvesting the differentiated cell from the enriched cellpopulation.

Another embodiment of the invention is a method of treating humanpluripotent stem (hPS) cells to obtain differentiated cells that can bemaintained in an in vitro culture, by providing a culture of the hPScells, and culturing the cells on a substrate in a culture mediumcontaining a hepatocyte differentiation agent under conditions thatpermit enrichment of the differentiated cells. Beneficial techniques andreagents for use in the context of such methods are detailed later inthe disclosure. Also embodied in the invention is a differentiated cellproduced according to a method of this invention, particularly thosehaving characteristics of cells of the hepatocyte lineage.

Yet another embodiment of the invention is a method of screening acompound for hepatocellular toxicity or modulation, comprisingcontacting a differentiated cell of this invention, and determining anyphenotypic or metabolic changes in the cell that result. Anotherembodiment of the invention is a method of detoxifying a fluid such asblood, comprising contacting a differentiated cell of this inventionwith the fluid under conditions that permit the cell to remove or modifya toxin in the fluid. In this context, the differentiated cellsdescribed in this disclosure can be used as part of a liver supportdevice, or for therapeutic administration for reconstitutinghepatocellular function in an individual.

These and other embodiments of the invention will be apparent from thedescription that follows.

DRAWINGS

FIG. 1 is a half-tone reproduction of a phase contrast photomicrograph(4×, 10×, 20×). Right side: Embryoid body cells from human pluripotentembryonic stem (hES) cells were cultured for 2 days in the hepatocytedifferentiating agent n-butyrate. The resulting cells show homogenousmorphology. Left side: Embryoid body cells cultured in serum (FBS)containing medium alone. There are heterogeneous patches of cells thatshow the morphology of many different cell types.

FIG. 2 is a half-tone reproduction of a phase contrast photomicrograph(10× in the upper two panels, 20× in the other panels). These are cellsthat have been differentiated by culturing 6 days with n-butyrate. Thecells predominantly demonstrate characteristics of mature hepatocytes.The cells in this field are binucleated and polygonal in shape, andexpress markers of mature hepatocytes detectable by immunostaining orreverse PCR.

FIG. 3 is a half-tone reproduction showing the results ofimmunohistochemical staining for certain cell specific markers (rightside), compared with the position of cell nuclei in the same field(bisbenzimide staining, left side). FIG. 3A (40×) shows the results foradult human hepatocytes; FIG. 3B (20×) shows the results for hES cellsdifferentiated by culturing 6 days with n-butyrate. Both cultures show ahigh proportion of cells staining positive for albumin, α₁-antitrypsin,and CD18, (three markers characteristic of cells of the hepatocytelineage), and negative for α-fetoprotein (a marker of less maturecells).

FIG. 4 is a half-tone reproduction of cells stained with Periodic AcidSchiff for the presence of glycogen (10× and 40×). ˜60% of the butyratetreated cells (top row) show evidence of glycogen storage, compared with˜80% in fetal hepatocytes (middle row) and virtually none in thefibroblast cell line (bottom row).

FIG. 5 is a half-tone reproduction of a phase contrast photomicrograph(10×, 40×), showing cells at various times during an exemplarydifferentiation and maturation process. Row A shows cells 4 days afterculture in SR medium containing 5 mM sodium n-butyrate. More than 80% ofcells in the culture are large in diameter, containing large nuclei andgranular cytoplasm. After 5 days, the cells were switched to specializedhepatocyte culture medium (HCM). Rows B and C show the appearance afterculturing in HCM for 2 or 4 days. Multinucleated polygonal cells arecommon. By these criteria, the ES-derived cells resemble freshlyisolated human adult hepatocytes (Row D) and fetal hepatocytes (Row E).

FIG. 6 is a bar graph, showing activity of cytochrome P-450 enzymes 1A1and 1A2 (CYP1A1/2). The enzyme was induced by culturing with 5 μmmethylchloranthrene (MC), and then measured using ethoxyresorufin.CYP1A1/2 activity was detected in two hepatocyte lineage lines derivedfrom the H1 line of ES cells, and one derived from the H9 line. Thelevel of activity exceeded the level observed in two preparations offreshly isolated human adult hepatocytes (HH). Activity inundifferentiated H1 and H9 cells and BJ embryonic fibroblasts wasnegligible.

DETAILED DESCRIPTION

This invention provides a system for preparing differentiated cells ofthe hepatocyte lineage from the pluripotent stem cells of primateorigin.

It has been discovered that when pluripotent stem cells are cultured inthe presence of a hepatocyte differentiation agent, a population ofcells is derived that has a remarkably high proportion of cells withphenotypic characteristics of cultured liver cells. Optionally, theeffect can be enhanced by also culturing the cells in the presence of ahepatocyte maturation factor. Since pluripotent stem cells canproliferate in culture for a year or more (over 300 populationdoublings), the invention described in this disclosure provides analmost limitless supply of hepatocyte-like cells, suitable for a varietyof developmental and therapeutic purposes.

FIG. 2 shows phase contrast photomicrographs of cells that have beendifferentiated by culturing with a prototype hepatocyte differentiationagent, n-butyrate. The cells show uniform features of hepatocytes,including a polygonal shape, and display characteristic phenotypicmarkers such as albumin, α₁-antitrypsin (AAT), and theasialoglycoprotein receptor, while lacking α-fetoprotein. The cells havebeen maintained in butyrate-containing medium for periods of 1-3 weeks.

The discovery is surprising, in view of the fact that histonedeacetylase inhibitors like butyrate and trichostatin A have beenimplicated in the differentiation of a wide variety of cell types. Apriori, it would be logical to predict that butyrate would drivepluripotent stem cell populations to differentiate into a widelyheterogeneous population, such as results from growing embryonic stemcells without feeders, or in the presence of retanoic acid. Contrary tothis prediction, a remarkably homogeneous population of hepatocytelineage cells is obtained.

This represents an important new paradigm in differentiation of humanpluripotent stem cell populations. To our knowledge, there have been nopublic reports of such a uniform population of hepatocyte lineage cellsbeing obtained from any type of embryonic stem cell.

The effects of butyrate on DNA synthesis and marker expression inprimary hepatocyte cultures have been studied by Gladhaug et al. (CancerRes. 48:6560, 1988), Engelmann et al. (In vitro Cell. Dev. Biol. 23:86,1987), Staecker et al. (J. Physiol. 135:367, 1988; Arch. Biochem.Biophys. 261:291, 1988; and Biochem. Biophys. Res. Commun. 147:78,1987). The effects of butyrate on human liver cell lines has beenstudied by Saito et al. (Int. J. Cancer 48:291, 1991) and Yoon et al.(Int. J. Artif. Organs 22:769, 1999). The effects of butyrate on ratoval cells (a hepatocyte precursor) have been studied by Pack et al.(Exp. Cell Res. 204:198, 1993), and Germain et al. (Cancer Res. 48:368,1988). The effect of Trichostatin A on rat hepatic stellate cells inprimary culture was studied by Niki et al. (Hepatology 29:858, 1999; andEuropean Patent Application EP 9837742 Al). The effect of butyrate onembryonic rat liver epithelial cells bipotential for hepatocytes andbiliary epithelium was studied by Blouin et al. (Exp. Cell Res. 21:22,1995). The effect of butyrate on cultured rat liver epithelial cellprecursors was studied by Coleman et al. (J. Cell. Physiol. 161:463,1994). L. E. Rogler (Am. J. Pathol. 150:591, 1997) reported thattreatment of a mouse hepatoblast cell line with DMSO or sodium butyrateinduced rapid hepatocytic differentiation. Watkins et al. (J. Dairy Res.66:559, 1999) report that butyric acid can also induce apoptosis inhuman hepatic tumor cells. All these studies relate to cells that aremature hepatocytes, either transformed liver cells, or committedhepatocyte precursor cells.

Butyrate has been shown to have a differentiating and modulating effecton a variety of other cell types, both in culture and in vivo. Kosugi etal. (Leukemia 13:1316, 1999) and Tamagawa (Biosci. Biotechnol. Biochem.62:1483, 1998) report that histone deacetylase inhibitors are potentinducers of differentiation in acute myeloid leukemia cells. Davis etal. (Biochem J. 346 pt 2:455, 2000) and Rivero et al. (Biochem. Biophys.Res. Commun. 248:664, 1998) discuss the effect of butyrate inerythroblastic differentiation. Perrine et al. (Am. J. Pediatr. Hematol.Oncol. 16:67, 1994) and Perrine et al. (N. Engl. J. Med. 328:81, 1993have proposed butyrate derivatives as agents for stimulating fetalglobin production in beta-globin disorders. Tai et al. (Hematol. Oncol.14:181, 1996) analyze the effect of butyrate differentiation ofeosinophilic granule-containing cells.

U.S. Pat. No. 5,763,255 report methods for inducing differentiation ofepithelial cells, in which 5 mM butyric acid is added toundifferentiated cells on a dried native fibrillar collagen cell culturesubstrate. Yamada et al. (Biosci. Biotech. Biochem. 56:1261, 1992)studied the effects of butyrate on three fibroblast and two epithelialcell lines. Jeng et al. (J. Periodontal. 70:1435, 1999) studied theeffects of butyrate and propionate on cultured gingival fibroblasts.Devereux et al. (Cancer Res. 59:6087, 1999) reported that treatment of ahuman fibroblast cell line with trichostatin A induced the cells toexpress telomerase reverse transcriptase. Yabushita et al. (Oncol. Res.5:173, 1993) studied the effects of butyrate, DMSO and dibutyryl cAMP onovarian adenocarcinoma cells. Graham et al. (J. Cellular Physiol.136:63, 1988) report that sodium butyrate induces differentiation ofbreast cancer cell lines. Kamitani (Arch. Biochem. Biophys. 368:45,1999), Siavoshian et al. (Gut 46:507, 2000), and Reynolds et al. (CancerLett. 11:53, 1998) studied the effect of sodium butyrate andtrichostatin A on the proliferation and differentiation of humanintestinal epithelial cells and colon cancer cells. McBain et al.(Biochem. Pharmacol. 53:1357, 1997) report that apoptotic death inadenocarcinoma cell lines can be induced by butyrate and other histonedeacetylase inhibitors.

Rocchi et al. (Anticancer Res. 18:1099, 1998) and Matsui et al. (BrainRes. 843:112, 1999) report the effect of butyrate analogues onproliferation, differentiation, and induction of catecholamine synthesisin human neuroblastoma cells. Gillenwater et al. (Head Neck 2:247, 2000)studied the effects of sodium butyrate on squamous carcinoma cell lines.Buommino et al. (J. Mol. Endocrinol.) studied the effect of butyrate oncell differentiation of seminal vesicle epithelial cells. Sun et al.(Lipids 32:273, 1997) studied butyrate-induced differentiation of gliomacells. Wang et al. (Exp. Cell. Res. 198:27, 1992) studied the effect ofn-butyrate in differentiating normal human keratinocytes. Perez et al.(J. Surg. Res. 78:1, 1998) report that butyrate upregulates PGE2production by Kupffer cells and modulates immune function. Schultz etal. (J. Exp. Zool. (Mol. Dev. Evol.) 285:276, 1999) found that treatmentof 2-cell embryos with histone deacetylase inhibitors reprogrammedexpression of certain genes. Chen et al. (Proc. Natl. Acad. Sci.94:5798,1997 and PCT application WO 97/47307) report the use of histonedeacetylase inhibitors for reactivating virally transduced genes. Simonet al. (Regul. Pept. 70:143, 1997) studied the effects of butyrate oninducing differentiation of pancreatic islet cells, resulting in anincrease in insulin production.

Because butyrate and related compounds promote differentiation in such alarge number of different cell types, one would expect a priori thattreating a mixed cell population derived from pluripotent embryoniccells would cause each cell in the population to differentiate furtheralong the line to which it is already committed—resulting simply in amore mature mixed cell population. It could not have been predicted thatbutyrate treatment would result in a uniform cell population—or whattissue type such cells would become.

This invention relates to the surprising discovery that culturingembryonic pluripotent cells with butyrate (or another hepatocytedifferentiation factor, detailed below) produces a population of cellsthat has a remarkably high proportion of cells with phenotypiccharacteristics of liver cells.

A frequent consequence of culturing pluripotent cells with thedifferentiation factors is that over 80% of cells are lost from theculture in the first 24 hours. What emerges after several days inculture is a population predominated by cells having characteristicfeatures of the hepatocyte lineage—such as a polygonal binucleatedphenotype, markers such as α₁-antitrypsin, and albumin, and expressionof metabolically important enzyme activity, such as the cytochrome p450enzymes 1A1 and 1A2. While not implying any limitation on the practiceof the invention, it is hypothesized that butyrate and otherdifferentiation factors either help induce cells to commit to thehepatocyte lineage—or preferentially promote survival of cells of thehepatocyte lineage—or have a combination of both these effects.

What follows is a further description of how this culture system can beemployed to generate hepatocyte lineage cells from pluripotent embryonicstem cells of primate origin. The use of hepatocyte differentiationagents (exemplified by but not limited to n-butyrate) is described,along with other features of the culture system that promote generationof hepatocyte lineage cells in culture.

Since pluripotent embryonic stem cells can essentially be grownindefinitely, this system provides an unbounded supply ofhepatocyte-like cells for use in research, pharmaceutical development,and the therapeutic management of liver disease.

Definitions

The terms “hepatocyte lineage” cell, “hepatoblastoid” cell and“hepatoembryoid” cell may be used in reference to the differentiatedcells of this invention, obtained by differentiating pluripotent cellsin the manner described. The differentiated cells have at least one of avariety of distinguishing phenotypic characteristics of known hepatocyteprecursor cells, hepatoblasts, and hepatocytes, as provided later inthis disclosure. By the use of these terms, no particular limitation isimplied with respect to cell phenotype, cellular markers, cell function,or proliferative capacity, except where explicitly required.

A “hepatocyte precursor cell” or a “hepatocyte stem cell” is a cell thatcan proliferate and further differentiate into a hepatocyte, undersuitable environmental conditions. Such cells may on occasion have thecapacity to produce other types of progeny, such as oval cells, bileduct epithelial cells, or additional hepatocyte precursor cells.

“Hepatocyte differentiation agent” and “hepatocyte maturation factor”are two terms with different meanings used in this disclosure torepresent a collection of compounds that can be used in preparing andmaintaining the differentiated cells of this invention. These agents arefurther described and exemplified in the sections that follow. The termsare not meant to imply a particular mode or timing of action, and nosuch limitation should be inferred. A “hepatocyte proliferative factor”is a biological or synthetic compound (a peptide, oligosaccharide, orthe like) that promotes the proliferation of hepatocytes and/orhepatocyte precursor cells.

Prototype “primate Pluripotent Stem cells” (pPS cells) are pluripotentcells derived from pre-embryonic, embryonic, or fetal tissue at any timeafter fertilization, and have the characteristic of being capable underthe right conditions of producing progeny of several different celltypes. As defined for the purposes of this disclosure, pPS cells arecapable of producing progeny that are derivatives of all of the threegerminal layers: endoderm, mesoderm, and ectoderm, according to astandard art-accepted test, such as the ability to form a teratoma in asuitable host.

Non-limiting exemplars of pPS cells are human embryonic stem (hES)cells, as described by Thomson et al., Science 282:1145, 1998; embryonicstem cells from other primates, such as Rhesus stem cells described byThomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995; and humanembryonic germ (hEG) cells, described in Shamblott et al., Proc. Natl.Acad. Sci. USA 95:13726, 1998. Other types of non-malignant pluripotentcells are also included in the term. Specifically, any cells of primateorigin that are fully pluripotent (capable of producing progeny that arederivatives of all three germinal layers) are included, regardless ofwhether they were derived from embryonic tissue, fetal tissue, or othersources.

pPS cell cultures are said to be “essentially undifferentiated” whenthey display the morphology that clearly distinguishes them fromdifferentiated cells of embryo or adult origin. pPS cells typically havehigh nuclear/cytoplasmic ratios, prominent nucleoli, and compact colonyformation with poorly discernable cell junctions, and are easilyrecognized by those skilled in the art. Colonies of undifferentiatedcells can be surrounded by neighboring cells that are differentiated.Nevertheless, the essentially undifferentiated colony will persist whencultured under appropriate conditions, and undifferentiated cellsconstitute a prominent proportion of cells proliferating upon passagingof the cultured cells. Cell populations that contain any proportion ofundifferentiated pPS with these criteria can be used in this invention.Cell cultures described as essentially undifferentiated will typicallycontain at least about 20%, 40%, 60%, or 80% undifferentiated pPS, inorder of increasing preference.

“Feeder cells” or “feeders” are terms used to describe cells of one typethat are co-cultured with cells of a second type, to provide anenvironment in which the cells of the second type can be maintained, andperhaps proliferate. The feeder cells are optionally from a differentspecies as the cells they are supporting. For example, certain pPS cellscan be supported by mouse embryonic fibroblasts (from primary culture ora telomerized line) or human fibroblast-like or mesenchymal cells (suchas can be differentiated and selected from hES cells). Typically (butnot necessarily), feeder cells are inactivated by irradiation ortreatment with an anti-mitotic agent such as mitomycin C, to preventthem from outgrowing the cells they are supporting.

pPS cell populations are said to be “essentially free” of feeder cellsif the cells have been passaged to a new culture environment withoutadding fresh feeder cells. It is recognized that if a previous culturecontaining feeder cells is used as a source of pPS for passaging, therewill be some feeder cells that survive the passage. For example, hEScells are often cultured in a 9.6 cm² well on a surface of 375,000primary irradiated embryonic fibroblasts near confluence. At the time ofthe next passage, perhaps 150,000 feeder cells are still viable, andwill be split and passaged along with hES that have proliferated to anumber of ˜1 to 1.5 million. After a 1:6 split, the hES cells generallyresume proliferation, but the fibroblasts will not grow and only a smallproportion will be viable by the end of ˜6 days of culture. This cultureis “essentially free” of feeder cells, with compositions containing lessthan about 5%, 1%, and 0.2% feeder cells being increasingly morepreferred.

A “growth environment” is an environment in which cells of interest willproliferate in vitro. Features of the environment include the medium inwhich the cells are cultured, the temperature, the partial pressure ofO₂ and CO₂, and a supporting structure (such as a substrate on a solidsurface) if present.

A “nutrient medium” is a medium for culturing cells containing nutrientsthat promote proliferation. The nutrient medium may contain any of thefollowing in an appropriate combination: isotonic saline, buffer, aminoacids, antibiotics, serum or serum replacement, and exogenously addedfactors.

A “conditioned medium” is prepared by culturing a first population ofcells in a medium, and then harvesting the medium. The conditionedmedium (along with anything secreted into the medium by the cells) maythen be used to support the growth of a second population of cells.

The term “antibody” as used in this disclosure refers to both polyclonaland monoclonal antibody. The ambit of the term deliberately encompassesnot only intact immunoglobulin molecules, but also such fragments andderivatives of immunoglobulin molecules (such as single chain Fvconstructs, diabodies, and fusion constructs) as may be prepared bytechniques known in the art, and retaining a desired antibody bindingspecificity.

“Restricted developmental lineage cells” are cells derived fromembryonic tissue, typically by differentiation of pPS cells. These cellsare capable of proliferating and may be able to differentiate intoseveral different cell types, but the range of phenotypes of theirprogeny is limited. Examples include: hematopoetic cells, which arepluripotent for blood cell types; neural precursors, which can generateglial cell precursors that progress to oligodendrocytes; neuronalrestrictive cells, which progress to various types of neurons; andhepatocyte progenitors, which are pluripotent for hepatocytes andsometimes other liver cells, such as bile duct epithelium.

General Techniques

For further elaboration of general techniques useful in the practice ofthis invention, the practitioner can refer to standard textbooks andreviews in cell biology, tissue culture, and embryology. Included areTeratocarcinomas and embryonic stem cells: A practical approach (E. J.Robertson, ed., IRL Press Ltd. 1987); Guide to Techniques in MouseDevelopment (P. M. Wasserman et al. eds., Academic Press 1993);Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles, Meth.Enzymol. 225:900, 1993); Properties and uses of Embryonic Stem Cells:Prospects for Application to Human Biology and Gene Therapy (P. D.Rathjen et al., Reprod. Fertil.

Dev. 10:31, 1998). General information and methodology relating to cellsof hepatocyte lineage is found in Liver Stem Cells (S. Sell & Z. Ilic,R. G. Landes Co., 1997), in Stem cell biology . . . (L. M. Reid, Curr.Opinion Cell Biol. 2:121, 1990), and in Liver Stem Cells (J. W. Grisham,pp 232-282 in Stem Cells, Academic Press, 1997). Use of hepatocyte-likecells in pharmaceutical research is described in In vitro Methods inPharmaceutical Research (Academic Press, 1997).

Methods in molecular genetics and genetic engineering are described inMolecular Cloning: A Laboratory Manual, 2nd Ed. (Sambrook et al., 1989);Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture(R. I. Freshney, ed., 1987); the series Methods in Enzymology (AcademicPress, Inc.); Gene Transfer Vectors for Mammalian Cells (I. M. Miller &M. P. Calos, eds., 1987); Current Protocols in Molecular Biology andShort Protocols in Molecular Biology, 3rd Edition (F. M. Ausubel et al.,eds., 1987 & 1995); and Recombinant DNA Methodology II (R. Wu ed.,Academic Press 1995). Reagents, cloning vectors, and kits for geneticmanipulation referred to in this disclosure are available fromcommercial vendors such as BioRad, Stratagene, Invitrogen, ClonTech, andSigma Chemical Co.

General techniques used in raising, purifying and modifying antibodies,and the design and execution of immunoassays includingimmunohistochemistry, the reader is referred to Handbook of ExperimentalImmunology (D. M. Weir & C. C. Blackwell, eds.); Current Protocols inImmunology (J. E. Coligan et al., eds., 1991); and R. Masseyeff, W. H.Albert, and N. A. Staines, eds., Methods of Immunological Analysis(Weinheim: VCH Verlags GmbH, 1993).

Media and Feeder Cells

Media for isolating and propagating pPS cells can have any of severaldifferent formulas, as long as the cells obtained have the desiredcharacteristics, and can be propagated further. Suitable sources are asfollows: Dulbecco's modified Eagle's medium (DMEM), Gibco #11965-092;Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco #10829-018;200 mM L-glutamine, Gibco #15039-027; non-essential amino acid solution,Gibco 11140-050; β-mercaptoethanol, Sigma #M7522; human recombinantbasic fibroblast growth factor (bFGF), Gibco #13256-029. Exemplaryserum-containing ES medium is made with 80% DMEM (typically KO DMEM),20% defined fetal bovine serum (FBS) not heat inactivated, 1%non-essential amino acids, 1 mM L-glutamine, and 0.1 mMβ-mercaptoethanol. Serum-free ES medium is made with 80% KO DMEM, 20%serum replacement, 1% non-essential amino acids, 1 mM L-glutamine, and0.1 mM β-mercaptoethanol. Not all serum replacements work, an effectiveserum replacement is Gibco #10828-028. Information on serum free mediain the propagation of pluripotent stem cells is published inInternational Patent Publications WO 97/47734 (Pedersen, U. California)and WO 98/30679 (Price et al., Life Technologies). The medium isfiltered and stored at 4° C. for no longer than 2 weeks. Just beforeuse, human bFGF is added to a final concentration of 4 ng/mL (Bodnar etal., Geron Corporation, International Patent Publication WO 99/20741).

pPS cells are typically cultured on a layer of feeder cells that supportthe pPS cells in various ways, such as the production of soluble factorsthat promote pPS cell survival or proliferation, or inhibitdifferentiation. Feeder cells are typically fibroblast type cells, oftenderived from embryonic or fetal tissue. A frequently used source offeeder fibroblasts is mouse embryo. The feeder cells are plated to nearconfluence, irradiated to prevent proliferation, and used to support pPScell cultures.

In an illustration of culturing pPS cells on feeder layers, mouseembryonic fibroblasts (mEF) are obtained from outbred CF1 mice (obtainedfrom SASCO) or other suitable strains. The abdomen of a mouse at 13 daysof pregnancy is swabbed with 70% ethanol, and the decidua is removedinto phosphate buffered saline (PBS). Embryos are harvested; placenta,membranes, and soft tissues are removed; and the carcasses are washedtwice in PBS. They are then transferred to fresh 10 cm culture dishescontaining 2 mL trypsin/EDTA, and finely minced. After incubating 5 minat 37° C., the trypsin is inactivated with 5 mL DMEM containing 10%fetal bovine serum (FBS), and the mixture is transferred to a 15 mLconical tube and dissociated. Debris is allowed to settle for 2 min, thesupernatant is made up to a final volume of 10 mL, and plated onto a 10cm tissue culture plate or T75 flask. The flask is incubated undisturbedfor 24 h, after which the medium is replaced. When flasks are confluent(˜1-2 d), the cells are split 1:2 into new flasks.

Feeder cells are propagated in mEF medium, containing 90% DMEM (Gibco#11965-092), 10% FBS (Hyclone #30071-03), and 2 mM glutamine. mEF arepropagated in T150 flasks (Corning #430825), splitting the cells 1:2every other day with trypsin, keeping the cells subconfluent, andoptionally frozen when necessary. To prepare the feeder cell layer,cells are irradiated at a dose to inhibit proliferation but permitsynthesis of important factors that support hES cells (˜4000 rads gammairradiation). Six-well culture plates (such as Falcon #304) are coatedby incubation at 37° C. with 1 mL 0.5% gelatin per well overnight, andplated with 375,000 irradiated mEF per well. Feeder cell layers are used5 h to 1 week after plating. The medium is replaced with fresh hESmedium just before seeding pPS cells.

Preparation of Primate Pluripotent Stem (pPS) Cells

Embryonic stem cells can be isolated from blastocysts of members of theprimate species (Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844,1995). Human embryonic stem (hES) cells can be prepared from humanblastocyst cells using the techniques described by Thomson et al. (U.S.Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133ff., 1998).

To obtain human blastocysts, human in vivo preimplantation embryos or invitro fertilized (IVF) embryos can be used or one cell human embryos canbe expanded to the blastocyst stage (Bongso et al., Hum Reprod 4: 706,1989). Briefly, human embryos are cultured to the blastocyst stage inG1.2 and G2.2 medium (Gardner et al., Fertil. Steril. 69:84, 1998).Blastocysts that develop are selected for ES cell isolation. The zonapellucida is removed from blastocysts by brief exposure to pronase(Sigma). The inner cell masses are isolated by immunosurgery, in whichblastocystsare exposed to a 1:50 dilution of rabbit anti-human spleencell antiserum for 30 minutes, then washed for 5 minutes three times inDMEM, and exposed to a 1:5 dilution of Guinea pig complement (Gibco) for3 minutes (see Solter et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975).After two further washes in DMEM, lysed trophectoderm cells are removedfrom the intact inner cell mass (ICM) by gentle pipetting, and the ICMplated on mEF.

After 9 to 15 days, inner cell mass-derived outgrowths are dissociatedinto clumps either by exposure to calcium and magnesium-freephosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to dispaseor trypsin, or by mechanical dissociation with a micropipette; and thenreplated on mEF in fresh medium. Dissociated cells are replated onembryonic feeder layers in fresh ES medium, and observed for colonyformation. Colonies demonstrating undifferentiated morphology areindividually selected by micropipette, mechanically dissociated intoclumps, and replated. ES-like morphology is characterized as compactcolonies with a high nucleus to cytoplasm ratio and prominent nucleoli.

Human Embryonic Germ (hEG) cells can be prepared from primordial germcells present in human fetal material taken about 8-11 weeks after thelast menstrual period. Suitable preparation methods are described inShamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 andInternational Patent Application WO 98/43679.

Briefly, genital ridges are rinsed with isotonic buffer, then placedinto 0.1 mL 0.05% trypsin-0.53 mM Sodium EDTA solution (BRL) and cutinto <1 mm³ chunks. The tissue is then pipetted through a 100 μL pipettip to further disaggregate the cells. It is incubated at 37° C. forapproximately 5 min, then approximately 3.5 mL EG growth medium isadded. EG growth medium is DMEM, 4500 mg/L D-glucose, 2200 mg/L mMsodium bicarbonate; 15% ES qualified fetal calf serum (BRL); 2 mMglutamine (BRL); 1 mM sodium pyruvate (BRL); 1000-2000 U/mL humanrecombinant leukemia inhibitory factor (LIF, Genzyme); 1-2 ng/ml humanrecombinant basic fibroblast growth factor (bFGF, Genzyme); and 10 μMforskolin (in 10% DMSO).

Ninety-six well tissue culture plates are prepared in advance with a subconfluent layer of feeder cells cultured for 3 days in a modified EGgrowth medium free of LIF, bFGF or Forskolin, then irradiated with 5000rad γ-irradiation. Suitable feeders are STO cells (ATCC Accession No.CRL 1503). ˜0.2 mL of the primary germ cell suspension is added to eachof the prepared wells. The first passage is conducted after 7-10 days inEG growth medium, transferring each well to 1 well of a 24-well culturedish previously prepared with irradiated STO mouse fibroblasts.

Undifferentiated pPS cells have characteristic morphological features,with high nuclear/cytoplasmic ratios, prominent nucleoli, and compactcolony formation with poorly discernable cell junctions. It is desirableto obtain cells that have a “normal karyotype”, which means that thecells are euploid, wherein all human chromosomes are present and are notnoticeably altered. This characteristic is also desirable in anydifferentiated cells that are subsequently derived and propagated.

Characteristic embryonic antigens can be identified byimmunohistochemistry or flow cytometry, using antibodies for SSEA 1,SSEA-3 and SSEA-4 (Developmental Studies Hybridoma Bank, NationalInstitute of Child Health and Human Development, Bethesda Md.), andTRA-1-60 and TRA-1-81 (Andrews et al., in Robertson E, ed.Teratocarcinomas and Embryonic Stem Cells. IRL Press, 207-246, 1987).The SSEA-1 marker is typically low or absent on hES cells, but presenton hEG cells. Differentiation of cells in vitro generally results in theloss of SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81, and increased expressionof SSEA-1. pPS cells can also be characterized by the presence ofalkaline phosphatase activity, which can be detected by developing fixedcells with Vector Red as a substrate (Vector Laboratories, BurlingameCalif.), and detecting red fluorescence of the product using a rhodaminefilter system.

Pluripotency of embryonic stem cells can be confirmed by injectingapproximately 0.5-10×10⁶ cells into the rear leg muscles of 8-12 weekold male SCID mice. The resulting tumors can be fixed in 4%paraformaldehyde and examined histologically after paraffin embedding at8-16 weeks of development. Teratomas develop that demonstrate at leastone cell type of each of the three germ layers, such as cartilage,smooth muscle, and striated muscle (for mesoderm); stratified squamousepithelium with hair follicles, neural tube with ventricular,intermediate, and mantle layers (for ectoderm); ciliated columnarepithelium and villi lined by absorptive enterocytes and mucus-secretinggoblet cells (for endoderm).

Propagation of pPS Cells

Embryonic stem cells can be cultured on layers of feeder cells in anutrient medium. The ES cells are routinely split every 1-2 weeks bybrief trypsinization, exposure to Dulbecco's PBS (without calcium ormagnesium and with 2 mM EDTA), exposure to Dispase or to Type IVCollagenase (1 mg/ml; Gibco) or by selection of individual colonies bymicropipette. Clump sizes of about 50 to 100 cells are optimal.Alternatively, after incubation with the protease, cultures can bescraped, dissociated into small clusters, and re-seeded onto freshfeeder cells at a split ratio of 1:3 to 1:30.

Embryonic germ cells can be cultured on feeder cells with dailyreplacement of growth medium until cells morphology consistent with EGcells are observed, typically, 7-30 days with 1 to 4 passages. The cellsmaintain their pluripotency through several months of culture.

International Patent Application WO 99/20741 describes methods andmaterials for growing pluripotent stem cells in the absence of feedercells, on an extracellular matrix with a nutrient medium. Suitable arefibroblast matrices prepared from lysed fibroblasts or isolated matrixcomponent from a number of sources. The nutrient medium may containsodium pyruvate, nucleosides, and one or more endogenously added growthfactors, such as bFGF, and may be conditioned by culturing withfibroblasts.

In the absence of feeder cells, suitable substrates for propagation ofpPS include extracellular matrix components, such as Matrigel® (BectonDickenson) or laminin. Matrigel® is a soluble preparation ofextracellular matrix from Engelbreth-Holm-Swarm tumor cells that gels atroom temperature to form a reconstituted basement membrane. To avoid theeffect of growth factors present in the membrane (such as IGF-1, TGF,and PDGF), Growth Factor Reduced Matrigel® is available. The criticalcomponents of the matrix can be identified by preparing an artificialmixture of all the components and leaving out components seriatim todetermine the effect. Other mixtures of extracellular matrix componentsmay also be suitable. Examples include collagen, fibronectin,proteoglycan, entactin, heparan sulfate, and the like, in variouscombinations.

The pluripotent cells are then plated onto the substrate in a suitabledistribution and in the presence of a medium that promotes cellsurvival, propagation, and retention of the desirable characteristics.All these characteristics benefit from careful attention to the seedingdistribution. One feature of the distribution is the plating density. Ithas been found that plating densities of at least about 15,500 cellscm⁻² promote survival and limit differentiation. Typically, a platingdensity of between about 90,000 cm⁻² and about 170,000 cm⁻² is used.

Another significant feature is the dispersion of cells. The propagationof mouse stem cells involves dispersing the cells into a single-cellsuspension (Robinson, Meth. Mol. Biol. 75:173, 1997 at page 177). Incontrast, the passage of pPS cells in the absence of feeders benefitsfrom preparing the pPS cells in small clusters. Typically, enzymaticdigestion is halted before cells become completely dispersed (say, ˜5min with collagenase IV). The plate is then scraped gently with apipette, and the cells are triturated with the pipette until they aresuspended as clumps of adherent cells, about 10-2000 cells in size. Theclumps are then plated directly onto the substrate without furtherdispersal.

It has also been found that pPS cells plated in the absence of freshfeeder cells benefit from being cultured in a nutrient medium. Themedium will generally contain the usual components to enhance cellsurvival, including isotonic buffer, essential minerals, and eitherserum or a serum replacement of some kind. Also beneficial is a mediumthat has been conditioned to supply some of the elements provided byfeeder cells. Conditioned medium can be prepared by culturing irradiatedprimary mouse embryonic fibroblasts (or another suitable cellpreparation) at a density of 5×10⁵ cells per 9.6 cm² well in a serumreplacement medium such as KO DMEM plus 20% serum replacement,containing 4 ng/mL basic fibroblast growth factor (bFGF). The culturesupematant is harvested after 1 day at 37° C., and typicallysupplemented with additional growth factors that benefit pPS cellculture. For hES, a growth factor like bFGF is often used. For hEG,culture medium may be supplemented with a growth factor like bFGF, aninducer of gp130, such as LIF or Oncostatin-M, and perhaps a factor thatelevates cyclic AMP levels, such as forskolin. Various types of pPScells may benefit from other factors in the medium.

Cell populations propagated by several of these techniques often remainessentially undifferentiated through multiple passages over a number ofmonths. It is recognized that during certain passages, some cells aroundthe periphery of colonies may differentiate (particularly when replatedas single cells, or when large clusters are allowed to form). However,cultures typically reestablish a larger proportion of undifferentiatedcells with characteristic morphology during the culture period.Optimally, the propagated cells will have a doubling time of no morethan about 20-40 hours.

Materials and Procedures for Differentiating pPS Cells

Differentiated cells of this invention can be made by culturing pPScells in the presence of a hepatocyte differentiation agent. Optionally,the cells are also cultured in the presence of a hepatocyte maturationfactor, either simultaneously or sequentially to when they are culturedwith the differentiation agent. The resulting cells have phenotypiccharacteristics of the hepatocyte lineage, as described in the sectionthat follows.

In certain embodiments of the invention, differentiation of the pPS isinitiated by first forming embryoid bodies. General principles inculturing embryoid bodies are reported in O'Shea, Anat. Rec. (New Anat.)257:323, 1999. pPS cells are cultured in a manner that permitsaggregates to form, for which many options are available: for example,by overgrowth of a donor pPS cell culture, or by culturing pPS cells inculture vessels having a substrate with low adhesion properties, such asmethyl cellulose. Embryoid bodies are readily recognizable by thoseskilled in the art, and can be readily harvested and transferred to anew culture environment. The embryoid bodies will typically have anendoderm exterior, and mesoderm and ectoderm interior.

As illustrated in the example section below, embryoid bodies can also bemade in suspension culture. pPS cells are harvested by brief collagenasedigestion, dissociated into clusters, and plated in non-adherent cellculture plates. The aggregates are fed every few days, and thenharvested after a suitable period, typically 4-8 days. The aggregatesare then plated on a substrate suitable for cells of the hepatocytelineage. Exemplary are Matrigel® (Becton Dickenson), more fullydescribed earlier, laminin, various types of collagen, and gelatin.Other artificial matrix components, and combinations may be used. Matrixcan also be produced by first culturing a matrix-producing cell line(such as a line of fibroblasts, endothelial cells, or mesenchymal stemcells), and then lysing and washing away cell debris in such a way thatthe matrix remains attached to the surface of the vessel. Dispersion ofcells from the embryoid bodies is not usually necessary; the embryoidbodies can be plated directly onto the matrix. The cells are thencultured in a medium that contains the hepatocyte differentiation agent.

In other embodiments of this invention, the pPS cells are combined withthe differentiation agent without forming substantial numbers ofembryoid bodies—i.e., by adding the agent to a standard pPS cell cultureat or before the time it reaches confluence, but before it begins toovergrow. This is referred to in this disclosure as the directdifferentiation method. It is generally advantageous (but not required)that the pPS cells are in a feeder-free culture. pPS cells can beharvested and plated onto a new substrate, and medium containing thedifferentiation agent can be added. Alternatively, if the pPS cells arealready being maintained on a matrix suitable for culture of thedifferentiated cells, then the differentiation agent can be addeddirectly to the pPS culture without replating. The cultures areinspected daily to determine whether confluence is reached. It has beenfound that the yield of hepatocyte lineage cells can be as much as3-fold higher when the differentiation agent is added just as the cellsreach confluence, rather than at ˜60-80% confluence.

Differentiation to the hepatocyte lineage is further promoted byproviding a substrate typical of the environment for hepatocytes invivo. For example, certain extracellular matrix components provide asuitable surface, such as Matrigel® (Becton Dickenson), laminin, ormatrix obtained from lysed cells. Another suitable substrate fordifferentiation of these cells is gelatin. The cells are cultured in anutrient medium that contains buffer, ionic strength, and nutrientsadequate to maintain the cells (see generally WO 99/20741). Optimizationof medium for particular cells is within purview of the skilledpractitioner, and is exemplified elsewhere in this disclosure.

The cells are maintained in the environment containing a suitablesubstrate and the hepatocyte differentiation agent for a period of timesufficient to permit enrichment of the differentiated cells from othercells—as may be determined empirically. For example, the first day ofculture with a differentiation agent such as n-butyrate leads to releaseof about 90% of cultured embryoid body derived cells from the substrateinto the medium. These cells are then removed when the medium is changedafter 24 h, and the surviving cells are cultured in fresh mediumcontaining n-butyrate.

After sufficient culture period, the remaining cells are considerablyenriched for those having characteristics of hepatocytes and/orhepatocyte progenitor cells. For the hepatocyte differentiation agentn-butyrate, the culture period is typically about 4-8 days, often about6 days. The reader is cautioned that prolonged culture in the presenceof some of the differentiation agents of this invention may besuboptimal for maximizing yield of hepatocyte lineage cells. Otherdifferentiation agents such as n-butyrate are tolerated on an ongoingbasis. Under these circumstances, it can be advantageous to keep theagent in the medium to maintain the full phenotype of the differentiatedcell. Without intending to be limited by theory, it is a hypothesis ofthis invention that hepatocyte differentiation agents such as n-butyratemay have two effects: first, to promote differentiation of pPS cellsdown the hepatocyte lineage, and second, to preferentially select cellsof this lineage for survival as the culture continues.

Suitable Differentiation Agents

n-Butyrate is a model hepatocyte differentiation agent, illustrated inthe examples that follow. Those skilled in the art will readilyrecognize that a number of homologs of n-butyrate can readily beidentified that have a similar effect, and can be used as substitutes inthe practice of this invention.

One class of homologs consists of other hydrocarbons that have similarstructural and physicochemical properties to those of n-butyrate. Someof such homologs are acidic hydrocarbons comprising 3-10 carbon atoms inbranched, straight-chain or cyclic form, and a conjugate base selectedfrom the group consisting of a carboxylate, a sulfonate, a phosphonate,and other proton donors. Suitable examples include but are not limitedto n-butyric acid, isobutyric acid, 2-butenoic acid, 3-butenoic acid,propanoic acid, propenoic acid, pentanoic acid, pentenoic acid, othershort-chain fatty acids that are either saturated or unsaturated, aminobutyric acid, phenyl butyric acid, phenyl propanoic acid, phenyl aceticacid, phenoxyacetic acid, cinnamic acid, and dimethylbutyrate. Also ofinterest is a hydrocarbon sulfonate or phosphonate that is isostericwith such compounds, particularly propanesulfonic acid andpropanephosphonic acid, which are isosteric to n-butyrate.

In the naming of such compounds, it is understood that all stereoisomersare included unless explicitly stated otherwise. Compounds with acidicgroups may be provided in the acidic form or as the conjugate base, withany acceptable opposing counter-ion. Since the use of sodium n-butyratewould increase the ionic strength of the environment it is used in, theaction of other agents may be augmented by providing a change in ionicstrength, by adding a salt, if necessary.

Another class of homologs are derivatives of butyrate and butyratehomologs, including conjugates with other molecules, such as aminoacids, monosaccharides, and other acceptable conjugate pairs. Many suchderivatives have been developed as butyrate prodrugs that aretransformed to the active form in vivo or in situ by the presence of asuitable converting enzyme—for example, a protease or a glycosidase. Byway of illustration, members of this class include arginine butyrate,lysine butyrate, other butyrate amides, glucose pentabutyrate,tributyrin, diacetone glucose butyrate, other butyrate saccharides,aminobutyric acid, isobutyramide, pivaloyloxymethyl butyrate,1-(2-hydroxyethyl)4-)1-oxobutyl)-piperazine butyrate, other piperazinederivatives of butyrate, and piracetam (2-oxo-1-pyrrolidine acetamide,Notropyl™), a cyclic derivative of gamma-amino butyrate.

A further class of homologs are inhibitors of histone deacetylase.Non-limiting examples include trichostatin A, 5-azacytidine, trapoxin A,oxamflatin, FR901228, cisplatin, and MS-27-275. The reader is alsoreferred to antiprotosoal cyclic tetrapeptides in U.S. Pat. No.5,922,837; antibacterial agents in U.S. Pat. No. 5,925,659; corepressorinhibitors in WO 99/23885; and cyclic peptide derivatives in WO99/11659. Methods to identify compounds with histone deacetylaseinhibitors can be identified by de-repression of hormone receptorcompounds (WO 98/48825).

The hepatocyte differentiation activity of n-butyrate may rely at leastin part on an ability to inhibit histone deacetylase. Assays for histonedeacetylase activity can be used as a preliminary screen to selectcandidates for other differentiation agents. Many such assays areavailable. For example, U.S. Pat. No. 5,922,837 (col. 3 ff.) describesan assay using tritiated N-desmethoxyapicidin and a parasite or chickliver S100 solution as a source of deacetylase activity. The candidatecompound is added to the reaction mixture, and tritium release ismeasured using a filter method. Nare et al. (Anal. Biochem. 267:390,1999) have developed a scintillation proximity assay using a peptidefrom histone H4, with lysine e-amino groups acetylated with tritium, andbound to an SPA bead that scintillates proportionately to the amount ofproximal tritium. Histone deacetylase activity (obtained from extractsof HeLa cell nuclei) releases the labeled acetyl groups and decreasesscintillation, and the presence of a deacetylase inhibitor maintainsscintillation. Hoffman et al. (Nucl. Acids Res. 27:2057, 1999) describesa non-isotopic assay for histone deacetylase activity. A fluorescentsubstrate has been developed that is an aminocoumarine derivative ofΩ-acetylated lysine. This permits quantitation of substrate in thenanomolar concentration range, which allows for high throughputscreening of histone deacetylase inhibitors.

A definitive test for a suitable differentiation agent is its ability totransform pPS cell cultures into cultures rd enriched for cells of thehepatocyte lineage, as described in this disclosure. Candidatecompounds, optionally prescreened according to one or more of theabove-listed criteria, are added to cultures of pPS cells or embryoidbodies in a manner similar to what is known to be effective forn-butyrate. Any compound that can at least promote differentiation ofpPS cells down the hepatocyte lineage, or preferentially permit thegrowth of hepatoblast-type cells, or preferentially remove cells ofother lineages, will be beneficial in deriving certain differentiatedcell populations embodied in this invention.

Following these guidelines, the ability of particular compound orcombination of compounds to act as hepatocyte differentiation agentscomprises culturing a population of substantially undifferentiated pPScells, or a mixed population of differentiated pPS cells (such as thoseobtained from embryoid bodies or by overgrowth of a pPS culture) in thepresence of the compound, and then determining the effect on cellmorphology, marker expression, enzymatic activity, proliferativecapacity, or other features of interest in relation to cells of thehepatocyte lineage. For optimum results, several concentrations of thetest compound are evaluated. A suitable base concentration may beisoosmolar or isotonic with effective butyrate concentrations, or haveequivalent inhibitory capacity of another histone deacetylase. Thecompound can then be tested over a range of about 1/10^(th) to 10 timesthe base concentration, or more, to determine if it has the desiredhepatocyte differentiation capacity.

A compound will be considered effective as a differentiation agent if itis capable of producing from a culture of pPS cells or embryoid bodycells a population of cells in which at least 40% of the cells have atleast three characteristics of hepatoblasts or hepatocytes. Agents thatproduce more uniform populations having a greater number of hepatocytecharacteristics are advantageous in some contexts. It is recognized thatagents producing less uniform or less mature hepatocyte populations mayalso be advantageous if the cells retain another desirable feature (suchas hardiness to manipulation, or proliferation capacity). As describedbelow, such cell populations can be further enriched for the desiredcell type by sorting or adsorption techniques.

Optional Use of Maturation Factors

Enrichment for differentiated cells using a hepatocyte differentiationagent can be supplemented, if desired, by the use of a separate compoundor mixture of compounds that act as hepatocyte maturation factors. Suchagents may augment the phenotype change promoted by the differentiationagent, or they may push the differentiation pathway further towards moremature cells, or they may help select for cells of the hepatocytelineage (for example, by preferentially supporting their survival), orthey may promote more rapid proliferation of cells with the desiredphenotype.

Once class of hepatocyte maturation factors are soluble growth factors(peptide hormones, cytokines, ligand-receptor complexes, and the like)that are capable of promoting the growth of cells of the hepatocytelineage. Such factors include but are not limited to epidermal growthfactor (EGF), insulin, TGF-α, TGF-β, fibroblast growth factor (FGF),heparin, hepatocyte growth factor (HGF), Oncostatin M in the presence ofdexamethazone, IL-1, IL-6, IGF-I, IGF-II, HBGF-1, and glucagon.

Another class of hepatocyte maturation factors are corticosteroids,particularly glucocorticoids. Such compounds are a steroid or steroidmimetic, and affects intermediary metabolism, especially promotion ofhepatic glycogen deposition, and inhibiting inflammation. Included arenaturally occurring hormones exemplified by cortisol, and syntheticglucocorticoids such as dexamethazone (U.S. Pat. No. 3,007,923) and itsderivatives, prednisone, methylprednisone, hydrocortisone, andtriamcinolone (U.S. Pat. No. 2,789,118) and its derivatives.

Another class of hepatocyte maturation factors are organic solvents likeDMSO. Alternatives with similar properties include but are not limitedto dimethylacetamide (DMA), hexmethylene bisacetamide, and otherpolymethylene bisacetamides. Solvents in this class are related, inpart, by the property of increasing membrane permeability of cells. Alsoof interest are solutes such as nicotinamide. Testing for whether acandidate compound acts as a hepatocyte maturation factor for thepurpose of this invention is performed empirically: pPS cultures aredifferentiated into cells of the hepatocyte lineage using a hepatocytedifferentiation agent described above, in combination with a modelhepatocyte differentiation agent, such as a growth factor or DMSO (thepositive control). In parallel, pPS are subjected to a similar protocolusing the same differentiation agent and the candidate maturationfactor. Resultant cells are then compared phenotypically to determinewhether the candidate agent has a similar effect to that of the positivecontrol.

In particular embodiments of this invention, the hepatocytedifferentiation agent and the hepatocyte maturation factor are usedsimultaneously or sequentially. In one illustration, newly platedembryoid bodies or feeder-free pPS cultures are placed in a mediumcontaining both n-butyrate and DMSO, and cultured for 4, 6, or 8 days,or until characteristic features appear, replacing the mediumperiodically (say, every 24 h) with fresh medium containing n-butyrateand DMSO. In another illustration, EB or pPS cultures are first culturedwith n-butyrate and DMSO for 4, 6, or 8 days, then the medium isexchanged for a hepatocyte-friendly medium containing a cocktail ofgrowth factors (perhaps in combination with n-butyrate) for long-termculture or assay.

Following these guidelines, the ability of particular compound orcombination of compounds to act as hepatocyte maturation factorscomprises culturing a population of cells previously treated with ahepatocyte differentiation agent in the presence of the compound, orincluding the compound in a culture of cells being treated with ahepatocyte differentiation factor. The effect of the compound on cellmorphology, marker expression, enzymatic activity, proliferativecapacity, or other features of interest is then determined in comparisonwith parallel cultures that did not include the candidate compound. Foroptimum results, several concentrations of the test compound areevaluated. A suitable base concentration for organic solvents may beisoosmolar or isotonic with effective DMSO concentrations. Suitable baseconcentrations for growth factors, cytokines, and other hormones may beconcentrations known to have similar growth-inducing or hormone activityin other systems. The test compound can then be tested over a range ofabout 1/10^(th) to 10 times the base concentration to determine if ithas the desired effect on hepatocyte-directed maturation of pPS cells.

Once cells of the desired phenotype are obtained, the cells can beharvested for any desired use. In certain differentiated cellpopulations of this invention, the cells are sufficiently uniform inphenotype that they can be harvested simply by releasing the cells fromthe substrate (e.g., using collagenase or by physical manipulation), andoptionally washing the cells free of debris. If desired, the harvestedcells can be further processed by positive selection for desiredfeatures, or negative selection for undesired features. For example,cells expressing surface markers or receptors can be positively ornegatively selected by incubating the population with an antibody orconjugate ligand, and then separating out the bound cells—for example,by labeled sorting techniques, or adsorption to a solid surface.Negative selection can also be performed by incubating the populationwith a cytolytic antibody specific for the undesired marker, in thepresence of complement.

If desired, harvested cells can be transferred into other cultureenvironments, such as those described elsewhere for the propagation ofother types of hepatocyte preparations. See, for example, U.S. Pat. Nos.5,030,105 and 5,576,207; EP Patent Application EP 953,633; Angelli etal., Histochem. J. 29:205, 1997; Gomez-Lechon et al., p.130 ff. in Invivo Methods in Pharmaceutical Research, Academic Press, 1997).

Characteristics of Differentiated Cells

Cells can be characterized according to a number of phenotypic criteria.The criteria include but are not limited to the detection orquantitation of expressed cell markers, and enzymatic activity, and thecharacterization of morphological features and intercellular signaling.

Certain differentiated pPS cells embodied in this invention havemorphological features characteristic of hepatocytes. The features arereadily appreciated by those skilled in evaluating such things, andinclude any or all of the following: a polygonal cell shape, abinucleate phenotype, the presence of rough endoplasmic reticulum forsynthesis of secreted protein, the presence of Golgi-endoplasmicreticulum lysosome complex for intracellular protein sorting, thepresence of peroxisomes and glycogen granules, relatively abundantmitochondria, and the ability to form tight intercellular junctionsresulting in creation of bile canalicular spaces. A number of thesefeatures present in a single cell is consistent with the cell being amember of the hepatocyte lineage. Unbiased determination of whethercells have morphologic features characteristic of hepatocytes can bemade by coding micrographs of differentiated pPS cells, adult or fetalhepatocytes, and one or more negative control cells, such as afibroblast, or RPE (Retinal pigment epithelial) cells—then evaluatingthe micrographs in a blinded fashion, and breaking the code to determineif the differentiated pPS cells are accurately identified.

Cells of this invention can also be characterized according to whetherthey express phenotypic markers characteristic of cells of thehepatocyte lineage. Cell markers useful in distinguishing liverprogenitors, hepatocytes, and biliary epithelium, are shown in Table 1(adapted from p 35 of Sell & Zoran, Liver Stem Cells, R.G. Landes Co.,TX, 1997; and Grisham et al., p 242 of “Stem Cells”, Academic Press,1997).

TABLE 1 Liver Cell Markers early hepato- biliary progenitors cytesepithelium albumin + + − α₁-antitrypsin + + − α-fetoprotein + fetal & −postnatal CEA − − +(?) γ-glutamyl tranpeptidase + fetal + GST-P +fetal + glucose-6- + + − phosphatase catalase − + − M2-PK + fetal + L-PK− + fetal p450 mono- + + − oxygenase p-glycoprotein ? canaliculi − CK7 −− + CK8 + + + CK14 + − − CK18 + + + CK19 −(+) − + CKX + − + BDS₇ + − +OV1 + − + OV6 − − + OC.1 − − + OC.2 + − + OC.3 + − + BD.1 + − + A6 + − +HBD.1 + + + H.2 − + − H.4 − + − H-4 ? + − H-6 − + − HES₆ − + − RL16/79 −postnatal − RL23/36 − + − BPC₅ + − − Vimentin − − fetal HepPar1 + + −Cell-CAM + + − 105 DPP IV + canaliculi + lectin binding + − + sitesblood group + − + antigens

It has been reported that hepatocyte differentiation requires thetranscription factor HNF4α (Li et al., Genes Dev. 14:464, 2000). Markersindependent of HNF-4α expression include α1-antitrypsin, α-fetoprotein,apoE, glucokinase, insulin growth factors 1 and 2, IGF-1 receptor,insulin receptor, and leptin. Markers dependent on HNF-4α expressioninclude albumin, apoAI, apoAII, apoB, apoCIII, apoCII, aldolase B,phenylalanine hydroxylase, L-type fatty acid binding protein,transferrin, retinol binding protein, and erythropoietin (EPO). Othermarkers of interest include those exemplified in Examples 1, 2, and 6,below.

Assessment of the level of expression of such markers can be determinedin comparison with other cells. Positive controls for the markers ofmature hepatocytes include adult hepatocytes of the species of interest,and established hepatocyte cell lines, such as the HepG2 line derivedfrom a hepatoblastoma reported in U.S. Pat. No. 5,290,684. The reader iscautioned that permanent cell lines such as HepG2 may be metabolicallyaltered, and fail to express certain characteristics of primaryhepatocytes such as cytochrome p450. Cultures of primary hepatocytes mayalso show decreased expression of some markers after prolonged culture.Negative controls include cells of a separate lineage, such as an adultfibroblast cell line, or retinal pigment epithelial (RPE) cells.Undifferentiated pPS cells are positive for some of the markers listedabove, but negative for markers of mature hepatocytes, as illustrated inthe examples below.

Tissue-specific protein and oligosaccharide determinants listed in thisdisclosure can be detected using any suitable immunologicaltechnique—such as flow immunocytochemistry for cell-surface markers,immunohistochemistry (for example, of fixed cells or tissue sections)for intracellular or cell-surface markers, Western blot analysis ofcellular extracts, and enzyme-linked immunoassay, for cellular extractsor products secreted into the medium. Expression of an antigen by a cellis said to be “antibody-detectable” if a significantly detectable amountof antibody will bind to the antigen in a standard immunocytochemistryor flow cytometry assay, optionally after fixation of the cells, andoptionally using a labeled secondary antibody or other conjugate (suchas a biotin-avidin conjugate) to amplify labeling.

The expression of tissue-specific markers can also be detected at themRNA level by Northern blot analysis, dot-blot hybridization analysis,or by reverse transcriptase initiated polymerase chain reaction (RT-PCR)using sequence-specific primers in standard amplification methods. SeeU.S. Pat. No. 5,843,780 for further details. Sequence data for theparticular markers listed in this disclosure can be obtained from publicdatabases such as GenBank (URL www.ncbi.nIm.nih.gov:80/entrez).Expression at the mRNA level is said to be “detectable” according to oneof the assays described in this disclosure if the performance of theassay on cell samples according to standard procedures in a typicalcontrolled experiment results in clearly discernable hybridization oramplification product. Expression of tissue-specific markers as detectedat the protein or mRNA level is considered positive if the level is atleast 2-fold, and preferably more than 10- or 50-fold above that of acontrol cell, such as an undifferentiated pPS cell, a fibroblast, orother unrelated cell type.

Cells can also be characterized according to whether they displayenzymatic activity that is characteristic of cells of the hepatocytelineage. For example, assays for glucose-6-phosphatase activity aredescribed by Bublitz (Mol Cell Biochem. 108:141, 1991); Yasmineh et al.(Clin. Biochem. 25:109, 1992); and Ockerman (Clin. Chim. Acta 17:201,1968). Assays for alkaline phosphatase (ALP) and 5-nucleotidase(5′-Nase) in liver cells are described by Shiojiri (J. Embryol. Exp.Morph.62:139, 1981). A number of laboratories that serve the research.and health care sectors provide assays for liver enzymes as a commercialservice.

Cytochrome p450 is a key catalytic component of the mono-oxygenasesystem. It constitutes a family of hemoproteins responsible for theoxidative metabolism of xenobiotics (administered drugs), and manyendogenous compounds. Different cytochromes present characteristic andoverlapping substrate specificity. Most of the biotransforming abilityis attributable by the cytochromes designated 1A2, 2A6, 2B6, 3A4,2C9-11, 2D6, and 2E1 (Gomes-Lechon et al., pp 129-153 in “In vitroMethods in Pharmaceutical Research,” Academic Press, 1997).

A number of assays are known in the art for measuring cytochrome p450enzyme activity. For example, cells can be contacted with anon-fluorescent substrate that is convertible to a fluorescent productby p450 activity, and then analyzed by fluorescence-activated cellcounting (U.S. Pat. No. 5,869,243). Specifically, the cells are washed,and then incubated with a solution of 10 μM/L5,6-methoxycarbonylfluorescein (Molecular Probes, Eugene Oreg.) for 15min at 37° C. in the dark. The cells are then washed, trypsinized fromthe culture plate, and analyzed for fluorescence emission at ˜520-560nm. A cell is said to have the enzyme activity assayed for if the levelof activity in a test cell is more than 2-fold, and preferably more than10- or 100-fold above that of a control cell, such as a fibroblast.

The expression of cytochrome p450 can also be measured at the proteinlevel, for example, using specific antibody in Western blots, or at themRNA level, using specific probes and primers in Northern blots orRT-PCR. See Borlakoglu et al., Int. J. Biochem. 25:1659, 1993.Particular activities of the p450 system can also be measured:7-ethoxycoumarin O-de-ethylase activity, aloxyresorufin O-de-alkylaseactivity, coumarin 7-hydroxylase activity, p-nitrophenol hydroxylaseactivity, testosterone hydroxylation, UDP-glucuronyltransferaseactivity, glutathione S-transferase activity, and others (reviewed inGomes-Lechon et al., pp 411-431 in “In vitro Methods in PharmaceuticalResearch,” Academic Press, 1997). The activity level can then becompared with the level in primary hepatocytes, as shown in Table 2.

TABLE 2 Drug Metabolizing Activities in 24-H Primary Cultured HumanHepatocytes Isozyme Reaction Activity P450† 65 ± 8  (n = 10) NADPH-Cc‡Cytochrome c oxidation 23 ± 2  (n = 10) CYP1A1/2d§ Aryl hydrocarbonhydroxylation  2.93 ± 0.99 (n = 7) 7-Ethoxyresorufin O-de-ethylation 3.09 ± 2.52  (n = 14) CYP2A6§ Coumarin 7-hydroxylation 137 ± 42 (n = 6)CYP2B6§ 7-Pentoxyresorufin O-depentylation  3.28 ± 1.76  (n = 10) PhaseI 7-Benzoxyresorufin O-debenzylation  1.38 ± 0.33 (n = 5) CYP2C9§4′-Diclofenac hydroxylation 317 ± 73 (n = 9) CYP2E1§ p-Nitrophenolhydroxylation  89 ± 42 (n = 6) Chlorzoxazone 6-hydroxylation 27 ± 3 (n =3) CYP3A3-5§ Testosterone 6β-hydroxylation  195 ± 122 (n = 7)Testosterone 2β-hydroxylation  61 ± 16 (n = 7) Testosterone15β-hydroxylation 12.4 ± 8.6 (n = 7) mEH§ Benzo(a)pyrene 7,8-oxidehydration 180 ± 72  (n = 10) Phase II UDPG-t‡ 4-Methylumbelliferoneconjugation  3.6 ± 0.4 (n = 5) GSH-t‡ 1-Chloro-2,4-dinitrobenzeneconjugation  301 ± 112 (n = 8) *Mean ± s.d. enzymatic activitydetermined in 24-h cultured human hepatocytes. †Cytochrome P450 contentis expressed as picomoles per milligram of cellular protein. ‡NADPH-C,UDPG-t and GSH-t activities are expressed as nanomoles per milligram perminute. §CYP enzymatic activities are expressed as picomoles permilligram per minute.

Assays are also available for enzymes involved in the conjugation,metabolism, or detoxification of small molecule drugs. For example,cells can be characterized by an ability to conjugate bilirubin, bileacids, and small molecule drugs, for excretion through the urinary orbiliary tract. Cells are contacted with a suitable substrate, incubatedfor a suitable period, and then the medium is analyzed (by GCMS or othersuitable technique) to determine whether conjugation product has beenformed. Drug metabolizing enzyme activities include de-ethylation,dealkylation, hydroxylation, demethylation, oxidation,glucuroconjugation, sulfoconjugation, glutathione conjugation, andN-acetyl transferase activity (A. Guillouzo, pp 411-431 in “In vitroMethods in Pharmaceutical Research,” Academic Press, 1997). Assaysinclude peenacetin de-ethylation, procainamide N-acetylation,paracetamol sulfoconjugation, and paracetamol glucuronidation (Chesne etal., pp 343-350 in “Liver Cells and Drugs”, A. Guillouzo ed. John UbbeyEurotext, London, 1988).

Cells of the hepatocyte lineage can also be evaluated on their abilityto store glycogen. A suitable assay uses Periodic Acid Schiff (PAS)stain, which does not react with mono- and disaccharides, but stainslong-chain polymers such as glycogen and dextran. PAS reaction providesquantitative estimations of complex carbohydrates as well as soluble andmembrane-bound carbohydrate compounds. Kirkeby et al. (Biochem. Biophys.Meth. 24:225, 1992) describe a quantitative PAS assay of carbohydratecompounds and detergents. van der Laarse et al. (Biotech Histochem.67:303, 1992) describe a microdensitometric histochemical assay forglycogen using the PAS reaction. Evidence of glycogen storage isdetermined if the cells are PAS-positive at a level that is at least2-fold, and preferably more than 10-fold above that of a control cell,such as a fibroblast. The cells can also be characterized by karyotypingaccording to standard methods.

pPS cells differentiated according to this invention can have a numberof the aforementioned features, including antibody-detectable expressionof α₁-antitrypsin (AAT) or albumin; absence of antibody-detectableexpression of α-fetoprotein; RT-PCR detectable expression ofasialoglycoprotein receptor (either the ASGR-1 or ASGR-2 isotype);evidence of glycogen storage; evidence of cytochrome p450 orglucose-6-phosphatase activity; and morphological featurescharacteristic of hepatocytes. The more of these features that arepresent in a particular cell, the more it can be characterized as a cellof the hepatocyte lineage. Cells having at least 2, 3, 5, 7, or 9 ofthese features are increasingly more preferred. In reference to aparticular cell population as may be present in a culture vessel or apreparation for administration, uniformity between cells in theexpression of these features is often advantageous. In thiscircumstance, populations in which at least about 40%, 60%, 80%, 90%,95%, or 98% of the cells have the desired features are increasingly morepreferred.

Other desirable features of differentiated cells of this invention arean ability to act as target cells in drug screening assays, and anability to reconstitute liver function, both in vivo, and as part of anextracorporeal device. These features are further described in sectionsthat follow.

Telomerization of Differentiated Cells

It is desirable that cells of the hepatocyte lineage have the ability toreplicate in certain drug screening and therapeutic applications. Thecells of this invention can optionally be telomerized to increase theirreplication potential, either before or after they progress torestricted developmental lineage cells or terminally differentiatedcells. pPS cells that are telomerized may be taken down thedifferentiation pathway described earlier; or differentiated cells canbe telomerized directly.

Before and after telomerization, telomerase activity and expression ofhTERT gene product can be determined using reagents and methods known inthe art. For example, pPS cells are evaluated for telomerase using TRAPactivity assay (Kim et al., Science 266:2011, 1997; Weinrich et al.,Nature Genetics 17:498, 1997). Expression of hTERT at the mRNA level isevaluated by RT-PCR.

Cells are telomerized by genetically altering them by transfection ortransduction with a suitable vector, homologous recombination, or otherappropriate technique, so that they express the telomerase catalyticcomponent (TERT). Particularly suitable is the catalytic component ofhuman telomerase (hTERT), provided in International Patent ApplicationWO 98/14592. For certain applications, species homologs like mouse TERT(WO 99/27113) can also be used. Transfection and expression oftelomerase in human cells is described in Bodnar et al., Science279:349, 1998 and Jiang et al., Nat. Genet. 21:111, 1999. In anotherexample, hTERT clones (WO 98/14592) are used as a source of hTERTencoding sequence, and spliced into an EcoRI site of a PBBS212 vectorunder control of the MPSV promoter, or into the EcoRI site ofcommercially available pBABE retrovirus vector, under control of the LTRpromoter. Differentiated or undifferentiated pPS cells are geneticallyaltered using vector containing supernatants over a 8-16 h period, andthen exchanged into growth medium for 1-2 days. Genetically alteredcells are selected using 0.5-2.5 μg/mL puromycin, and recultured. Theycan then be assessed for hTERT expression by RT-PCR, telomerase activity(TRAP assay), immunocytochemical staining for hTERT, or replicativecapacity. Continuously replicating colonies will be enriched by furtherculturing under conditions that support proliferation, and cells withdesirable phenotypes can optionally be cloned by limiting dilution.

In certain embodiments of this invention, pPS cells are differentiatedinto cells bearing characteristics of the hepatocyte lineage, and thenthe differentiated cells are genetically altered to express TERT. Inother embodiments of this invention, pPS cells are genetically alteredto express TERT, and then differentiated into cells bearingcharacteristics of the hepatocyte lineage. Successful modification toincrease TERT expression can be determined by TRAP assay, or bydetermining whether the replicative capacity of the cells has improved.

Other methods of immortalizing cells are also contemplated, such astransforming the cells with DNA encoding the SV40 large T antigen (U.S.Pat. No. 5,869,243, International Patent Application WO 97/32972).Transfection with oncogenes or oncovirus products is less suitable whenthe cells are to be used for therapeutic purposes. Telomerized cells areof particular interest in applications of this invention where it isadvantageous to have cells that can proliferate and maintain theirkaryotype—for example, in pharmaceutical screening, and in therapeuticprotocols where differentiated cells are administered to an individualin order to augment liver function.

Use of Differentiated Cells

This invention provides a method by which large numbers of cells of thehepatocyte lineage can be produced. These cell populations can be usedfor a number of important research, development, and commercialpurposes.

Preparation of Expression Libraries and Specific Antibody

The differentiated cells of this invention can also be used to prepare acDNA library relatively uncontaminated with cDNA preferentiallyexpressed in cells from other lineages. For example, the cells arecollected by centrifugation at 1000 rpm for 5 min, and then mRNA isprepared from the pellet by standard techniques (Sambrook et al.,supra). After reverse transcribing into cDNA, the preparation can besubtracted with cDNA from any or all of the following cell types:undifferentiated pPS, embryonic fibroblasts, visceral endoderm,sinusoidal endothelial cells, bile duct epithelium, or other cells ofundesired specificity, thereby producing a select cDNA library,reflecting expression patterns that are representative of maturehepatocytes, hepatocyte precursors, or both.

The differentiated cells of this invention can also be used to prepareantibodies that are specific for hepatocyte markers, progenitor cellmarkers, markers that are specific for hepatocyte precursors, and otherantigens that may be expressed on the cells. The cells of this inventionprovide an improved way of raising such antibodies because they arerelatively enriched for particular cell types compared with pPS cellcultures and hepatocyte cultures made from liver tissue. Polyclonalantibodies can be prepared by injecting a vertebrate with cells of thisinvention in an immunogenic form. Production of monoclonal antibodies isdescribed in such standard references as Harrow & Lane (1988), U.S. Pat.Nos. 4,491,632, 4,472,500 and 4,444,887, and Methods in Enzymology 73B:3(1981). Other methods of obtaining specific antibody molecules(optimally in the form of single-chain variable regions) involvecontacting a library of immunocompetent cells or viral particles withthe target antigen, and growing out positively selected clones. SeeMarks et al., New Eng. J. Med. 335:730, 1996, International PatentApplications WO 94/13804, WO 92/01047, WO 90/02809, and McGuiness etal., Nature Biotechnol. 14:1449, 1996. By positively selecting using pPSof this invention, and negatively selecting using cells bearing morebroadly distributed antigens (such as differentiated embryonic cells) oradult-derived stem cells, the desired specificity can be obtained. Theantibodies in turn can be used to identify or rescue hepatocyteprecursor cells of a desired phenotype from a mixed cell population, forpurposes such as costaining during immunodiagnosis using tissue samples,and isolating such cells from mature hepatocytes or cells of otherlineages.

Genomics

Differentiated pPS cells are of interest to identify expression patternsof transcripts and newly synthesized proteins that are characteristicfor hepatocyte precursor cells, and may assist in directing thedifferentiation pathway or facilitating interaction between cells.Expression patterns of the differentiated cells are obtained andcompared with control cell lines, such as undifferentiated pPS cells,other types of committed precursor cells (such as pPS cellsdifferentiated towards other lineages, hematopoietic stem cells,precursor cells for other mesoderm-derived tissue, precursor cells forendothelium or bile duct epithelium, hepatocyte stem cells obtained fromadult tissues, or pPS cells differentiated towards the hepatocytelineage using alternative reagents or techniques).

Suitable methods for comparing expression at the protein level includethe immunoassay or immunohistochemistry techniques describe earlier.Suitable methods for comparing expression at the level of transcriptioninclude methods of differential display of mRNA (Liang, Peng, et al.,Cancer Res. 52:6966, 1992), and matrix array expression systems (Schenaet al., Science 270:467, 1995; Eisen et al., Methods Enzymol. 303:179,1999; Brown et al., Nat. Genet. 21 Suppl 1:33, 1999).

The use of microarray in analyzing gene expression is reviewed by Fritzet al Science 288:316, 2000; “Microarray Biochip Technology”, M. Schenaed., Eaton Publishing Company; “Microarray analysis”, Gwynne & Page,Science (Aug. 6, 1999 supplement); Pollack et al., Nat Genet 23:41,1999; Gerhold et al., Trends Biochem. Sci. 24:168, 1999; “Gene Chips(DNA Microarrays)”, L Shi, www.Gene-Chips.com. Systems and reagents forperforming microarray analysis are available commercially from companiessuch as Affymetrix, Inc., Santa Clara Calif.; Gene Logic Inc., ColumbiaMd.; Hyseq Inc., Sunnyvale Calif.; Molecular Dynamics Inc., SunnyvaleCalif.; Nanogen, San Diego Calif.; and Synteni Inc., Fremont Calif.(acquired by Incyte Genomics, Palo Alto Calif.).

Solid-phase arrays are manufactured by attaching the probe at specificsites either by synthesizing the probe at the desired position, or bypresynthesizing the probe fragment and then attaching it to the solidsupport. A variety of solid supports can be used, including glasses,plastics, ceramics, metals, gels, membranes, paper, and beads of variouscomposition. U.S. Pat. No. 5,445,934 discloses a method of on-chipsynthesis, in which a glass slide is derivatized with a chemical speciescontaining a photo-cleavable protecting group. Each site is sequentiallydeprotected by irradiation through a mask, and then reacted with a DNAmonomer containing a photoprotective group. Methods for attaching apresynthesized probe onto a solid support include adsorption, ultraviolet linking, and covalent attachment. In one example, the solidsupport is modified to carry an active group, such as hydroxyl,carboxyl, amine, aldehyde, hydrazine, epoxide, bromoacetyl, maleimide,or thiol groups through which the probe is attached (U.S. Pat. Nos.5,474,895 and 5,514,785).

The probing assay is typically conducted by contacting the array by afluid potentially containing the nucleotide sequences of interest undersuitable conditions for hybridization, and then determining any hybridformed. For example, mRNA or DNA in the sample is amplified in thepresence of nucleotides attached to a suitable label, such as thefluorescent labels Cy3 or Cy5. Conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of homology, as appropriate. The array is then washed, and boundnucleic acid is determined by measuring the presence or amount of labelassociated with the solid phase. Different samples can be comparedbetween arrays for relative levels of expression, optionallystandardized using genies expressed in most cells of interest, such as aribosomal or house-keeping gene, or as a proportion of totalpolynucleotide in the sample. Alternatively, samples from two or moredifferent sources can be tested simultaneously on the same array, bypreparing the amplified polynucleotide from each source with a differentlabel.

An exemplary method is conducted using a Genetic Microsystems arraygenerator, and an Axon GenePix™ Scanner. Microarrays are prepared byfirst amplifying cDNA fragments encoding marker sequences to be analyzedin a 96 or 384 well format. The cDNA is then spotted directly onto glassslides at a density as high as >5,000 per slide. To compare mRNApreparations from two cells of interest, one preparation is convertedinto Cy3-labeled cDNA, while the other is converted into Cy5-labeledcDNA. The two cDNA preparations are hybridized simultaneously to themicroarray slide, and then washed to eliminate non-specific binding. Anygiven spot on the array will bind each of the cDNA products inproportion to abundance of the transcript in the two original mRNApreparations. The slide is then scanned at wavelengths appropriate foreach of the labels, the resulting fluorescence is quantified, and theresults are formatted to give an indication of the relative abundance ofmRNA for each marker on the array.

Identifying expression products for use in characterizing and affectingdifferentiated cells of this invention involves analyzing the expressionlevel of RNA, protein, or other gene product in a first cell type, suchas a pPS cell differentiated along the hepatocyte lineage, analyzing theexpression level of the same product in a control cell type, comparingthe relative expression level between the two cell types, (typicallynormalized by total protein or RNA in the sample, or in comparison withanother gene product expected to be expressed at a similar level in bothcell types, such as a house-keeping gene), and identifying products ofinterest based on the comparative expression level.

Products will typically be of interest if their relative expressionlevel is at least about 2-fold, 10-fold, or 100-fold elevated (orsuppressed) in differentiated pPS cells of this invention, in comparisonwith the control. This analysis can optionally be computer-assisted, bymarking the expression level in each cell type on an independent axis,wherein the position of the mark relative to each axis is in accordancewith the expression level in the respective cell, and then selecting aproduct of interest based on the position of the mark. Alternatively,the difference in expression between the first cell and the control cellcan be represented on a color spectrum (for example, where yellowrepresents equivalent expression levels, red indicates augmentedexpression and blue represents suppressed expression). The product ofinterest can then be selected based on the color representing expressionof one marker of interest, or based on a pattern of colors representinga plurality of markers.

Differentiated pPS Cells for Drug Screening

Differentiated pPS cells of this invention can be used to screen forfactors (such as solvents, small molecule drugs, peptides,polynucleotides, and the like) or environmental conditions (such asculture conditions or manipulation) that affect the characteristics ofdifferentiated cells of the hepatocyte lineage.

In some applications, pPS cells (differentiated or undifferentiated) areused to screen factors that promote maturation of cells along thehepatocyte lineage, or promote proliferation and maintenance of suchcells in long-term culture. For example, candidate hepatocyte maturationfactors or growth factors are tested by adding them to pPS cells indifferent wells, and then determining any phenotypic change thatresults, according to desirable criteria for further culture and use ofthe cells.

Particular screening applications of this invention relate to thetesting of pharmaceutical compounds in drug research. The reader isreferred generally to the standard textbook “In vitro Methods inPharmaceutical Research”, Academic Press, 1997, and U.S. Pat. No.5,030,015). In this invention, pPS cells that have differentiated to thehepatocyte lineage play the role of test cells for standard drugscreening and toxicity assays, as have been previously performed onhepatocyte cell lines or primary hepatocytes in short-term culture.Assessment of the activity of candidate pharmaceutical compoundsgenerally involves combining the differentiated cells of this inventionwith the candidate compound, determining any change in the morphology,marker phenotype, or metabolic activity of the cells that isattributable to the compound (compared with untreated cells or cellstreated with an inert compound), and then correlating the effect of thecompound with the observed change. The screening may be done eitherbecause the compound is designed to have a pharmacological effect onliver cells, or because a compound designed to have effects elsewheremay have unintended hepatic side effects. Two or more drugs can betested in combination (by combining with the cells either simultaneouslyor sequentially), to detect possible drug-drug interaction effects.

In some applications, compounds are screened initially for potentialhepatotoxicity (Castell et al., pp 375-410 in “In vitro Methods inPharmaceutical Research,” Academic Press, 1997). Cytotoxicity can bedetermined in the first instance by the effect on cell viability,survival, morphology, and leakage of enzymes into the culture medium.More detailed analysis is conducted to determine whether compoundsaffect cell function (such as gluconeogenesis, ureogenesis, and plasmaprotein synthesis) without causing toxicity. Lactate dehydrogenase (LDH)is a good marker because the hepatic isoenzyme (type V) is stable inculture conditions, allowing reproducible measurements in culturesupernatants after 12-24 h incubation. Leakage of enzymes such asmitochondrial glutamate oxaloacetate transaminase and glutamate pyruvatetransaminase can also be used. Gomez-Lechon et al. (Anal. Biochem.236:296, 1996) describe a microassay for measuring glycogen, which canbe applied to measure the effect of pharmaceutical compounds onhepatocyte gluconeogenesis.

Other current methods to evaluate hepatotoxicity include determinationof the synthesis and secretion of w albumin, cholesterol, andlipoproteins; transport of conjugated bile acids and bilirubin;ureagenesis; cytochrome p450 levels and activities; glutathione levels;release of a-glutathione s-transferase; ATP, ADP, and AMP metabolism;intracellular K⁺ and Ca²⁺ concentrations; the release of nuclear matrixproteins or oligonucleosomes; and induction of apoptosis (indicated bycell rounding, condensation of chromatin, and nuclear fragmentation).DNA synthesis can be measured as [³H]-thymidine or BrdU incorporation.Effects of a drug on DNA synthesis or structure can be determined bymeasuring DNA synthesis or repair. [³H]-thymidine or BrdU incorporation,especially at unscheduled times in the cell cycle, or above the levelrequired for cell replication, is consistent with a drug effect.Unwanted effects can also include unusual rates of sister chromatidexchange, determined by metaphase spread. The reader is referred to A.Vickers (pp 375-410 in “In vitro Methods in Pharmaceutical Research,”Academic Press, 1997) for further elaboration.

Restoration of Liver Function

This invention also provides for the use of differentiated pPS cells torestore a degree of liver function to a subject needing such therapy,perhaps due to an acute, chronic, or inherited impairment of liverfunction.

To determine the suitability of differentiated pPS cells for therapeuticapplications, the cells can first be tested in a suitable animal model.At one level, cells are assessed for their ability to survive andmaintain their phenotype in vivo. Differentiated pPS cells areadministered to immunodeficient animals (such as SCID mice, or animalsrendered immunodeficient chemically or by irradiation) at a siteamenable for further observation, such as under the kidney capsule, intothe spleen, or into a liver lobule. Tissues are harvested after a periodof a few days to several weeks or more, and assessed as to whether pPScells are still present. This can be performed by providing theadministered cells with a detectable label (such as green fluorescentprotein, or β-galactosidase); or by measuring a constitutive markerspecific for the administered cells. Where differentiated pPS cells arebeing tested in a rodent model, the presence and phenotype of theadministered cells can be assessed by immunohistochemistry or ELISAusing human-specific antibody, or by RT-PCR analysis using primers andhybridization conditions that cause amplification to be specific forhuman polynucleotide sequences. Suitable markers for assessing geneexpression at the mRNA or protein level are provided in Table 3. Generaldescriptions for determining the fate of hepatocyte-like cells in animalmodels is provided in Grompe et al. (Sem. Liver Dis. 19:7, 1999);Peeters et al., (Hepatology 25:884, 1997;) and Ohashi et al. (NatureMed. 6:327, 2000).

At another level, differentiated pPS cells are assessed for theirability to restore liver function in an animal lacking full liverfunction. Braun et al. (Nature Med. 6:320, 2000) outline a model fortoxin-induced liver disease in mice transgenic for the HSV tk gene. Rhimet al. (Proc. Natl. Acad. Sci. USA 92:4942, 1995) and Lieber et al.(Proc. Natl. Acad. Sci. USA 92:6210, 1995) outline models for liverdisease by expression of urokinase. Mignon et al. (Nature Med. 4:1185,1998) outline liver disease induced by antibody to the cell-surfacemarker Fas. Overturf et al. (Human Gene Ther. 9:295, 1998) havedeveloped a model for Hereditary Tyrosinemia Type I in mice by targeteddisruption of the Fah gene. The animals can be rescued from thedeficiency by providing a supply of2-(2-nitro-4-fluoro-methyl-benzyol)-1,3-cyclohexanedione (NTBC), butdevelop liver disease when NTBC is withdrawn. Acute liver disease can bemodeled by 90% hepatectomy (Kobayashi et al., Science 287:1258, 2000).Acute liver disease can also be modeled by treating animals with ahepatotoxin such as galactosamine, CCl₄, or thioacetamide. Chronic liverdiseases such as cirrhosis can be modeled by treating animals with asub-lethal dose of a hepatotoxin long enough to induce fibrosis (Rudolphet al., Science 287:1253, 2000). Assessing the ability of differentiatedcells to reconstitute liver function involves administering the cells tosuch animals, and then determining survival over a 1 to 8 week period ormore, while monitoring the animals for progress of the condition.Effects on hepatic function can be determined by evaluating markersexpressed in liver tissue, cytochrome p450 activity, and bloodindicators, such as alkaline phosphatase activity, bilirubinconjugation, and prothrombin time), and survival of the host Anyimprovement in survival, disease progression, or maintenance of hepaticfunction according to any of these criteria relates to effectiveness ofthe therapy, and can lead to further optimization.

This invention includes differentiated cells that are encapsulated, orpart of a bioartificial liver device. Various forms of encapsulation aredescribed in “Cell Encapsulation Technology and Therapeutics”,Kuhtreiber et al. eds., Birkhauser, Boston Mass., 1999. Differentiatedcells of this invention can be encapsulated according to such methodsfor use either in vitro or in vivo.

Bioartificial organs for clinical use are designed to support anindividual with impaired liver function—either as a part of long-termtherapy, or to bridge the time between a fulminant hepatic failure andhepatic reconstitution or liver transplant. Bioartificial liver devicesare reviewed by Macdonald et al., pp. 252-286 of “Cell EncapsulationTechnology and Therapeutics”, op cit., and exemplified in U.S. Pat. Nos.5,290,684, 5,624,840, 5,837,234, 5,853,717, and 5,935,849.Suspension-type bioartificial livers comprise cells suspended in platedialysers, or microencapsulated in a suitable substrate, or attached tomicrocarrier beads coated with extracellular matrix. Alternatively,hepatocytes can be placed on a solid support in a packed bed, in amultiplate flat bed, on a microchannel screen, or surrounding hollowfiber capillaries. The device has inlet and outlet through which thesubject's blood is passed, and sometimes a separate set of ports forsupplying nutrients to the cells.

Current proposals for such liver support devices involve hepatocytesfrom a xenogeneic source, such as a suspension of porcine hepatocytes,because of the paucity of available primary human hepatocytes.Xenogeneic tissue sources raise regulatory concerns regardingimmunogenicity and possible cross-species viral transmission.

The present invention provides a system for generating preparativecultures of human cells. Differentiated pluripotent stem cells areprepared according to the methods described earlier, and then platedinto the device on a suitable substrate, such as a matrix of Matrigel®or collagen. The efficacy of the device can be assessed by comparing thecomposition of blood in the afferent channel with that in the efferentchannel—in terms of metabolites removed from the afferent flow, andnewly synthesized proteins in the efferent flow.

Devices of this kind can be used to detoxify a fluid such as blood,wherein the fluid comes into contact with the differentiated cells ofthis invention under conditions that permit the cell to remove or modifya toxin in the fluid. The detoxification will involve removing oraltering at least one ligand, metabolite, or other compound (eithernatural and synthetic) that is usually processed by the liver. Suchcompounds include but are not limited to bilirubin, bile acids, urea,heme, lipoprotein, carbohydrates, transferrin, hemopexin,asialoglycoproteins, hormones like insulin and glucagon, and a varietyof small molecule drugs. The device can also be used to enrich theefferent fluid with synthesized proteins such as albumin, acute phasereactants, and unloaded carrier proteins. The device can be optimized sothat a variety of these functions are performed, thereby restoring asmany hepatic functions as are needed. In the context of therapeuticcare, the device processes blood flowing from a patient in hepatocytefailure, and then the blood is returned to the patient.

Differentiated pPS cells of this invention that demonstrate desirablefunctional characteristics in animal models (such as those describedabove) may also be suitable for direct administration to human subjectswith impaired liver function. For purposes of hemostasis, the cells canbe administered at any site that has adequate access to the circulation,typically within the abdominal cavity. For some metabolic anddetoxification functions, it is advantageous for the cells to haveaccess to the biliary tract. Accordingly, the cells are administerednear the liver (e.g., in the treatment of chronic liver disease) or thespleen (e.g., in the treatment of fulminant hepatic failure). In onemethod, the cells administered into the hepatic circulation eitherthrough the hepatic artery, or through the portal vein, by infusionthrough an in-dwelling catheter. A catheter in the portal vein can bemanipulated so that the cells flow principally into the spleen, or theliver, or a combination of both. In another method, the cells areadministered by placing a bolus in a cavity near the target organ,typically in an excipient or matrix that will keep the bolus in place.In another method, the cells are injected directly into a lobe of theliver or the spleen.

The differentiated cells of this invention can be used for therapy ofany subject in need of having hepatic function restored or supplemented.Human conditions that may be appropriate for such therapy includefulminant hepatic failure due to any cause, viral hepatitis,drug-induced liver injury, cirrhosis, inherited hepatic insufficiency(such as Wilson's disease, Gilbert's syndrome, or al-antitrypsindeficiency), hepatobiliary carcinoma, autoimmune liver disease (such asautoimmune chronic hepatitis or primary biliary cirrhosis), and anyother condition that results in impaired hepatic function. For humantherapy, the dose is generally between about 10⁹ and 10¹² cells, andtypically between about 5×10⁹ and 5×10¹⁰ cells, making adjustments forthe body weight of the subject, nature and severity of the affliction,and the replicative capacity of the administered cells. The ultimateresponsibility for determining the mode of treatment and the appropriatedose lies with the managing clinician.

The following examples provided as further non-limiting illustrations ofparticular embodiments of the invention.

EXAMPLES Experimental Procedures

This section provides details of some of the techniques and reagentsused in the Examples below.

Maintenance of Human Embryonic Stem Cells:

hES cells were maintained on primary mouse embryonic fibroblasts inserum-free media. The hES cells were seeded as small clusters onirradiated mouse embryonic fibroblasts at about 40,000 cells cm⁻². Thesecultures were maintained in a medium composed of 80% KO DMEM (Gibco) and20% Serum Replacement (Gibco), supplemented with 1% non-essential aminoacids, 1 mM glutamine, :0.1 mM β-mercaptoethanol and 4 ng/mL human bFGF(Gibco). Cells were expanded by serial passaging of the ES colonies.This was accomplished by treating the monolayer culture of ES colonieswith 1 mg/mL collagenase for 5-20 minutes at 37° C. The cultures werethen gently scraped to remove the cells. The clusters were gentlydissociated, and replated as small clusters onto fresh feeder cells.

Production of Embtyoid Bodies (EB):

Confluent monolayer cultures of hES cells on or off feeder cells wereharvested by incubating in collagenase for 15-20 min, following whichthe cells are scraped from the plate. The cells were then issociatedinto clusters and plated in non-adherent cell culture plates (Costar) ina medium composed of 80% KO DMEM (Gibco) and 20% non-heat-inactivatedFBS (Hyclone), supplemented with 1% non-essential amino acids, 1 mMglutamine, 0.1 mM β-mercaptoethanol. The cells were seeded at a 1:2ratio in 2 mL medium per well (6 well plate). The EBs were fed everyother day by the addition of 2 mL of medium per well. When the volume ofmedium exceeded 4 mL/well, the EBs were collected and resuspended infresh medium. After 4-8 days in suspension, the EBs were plated onto asubstrate and allowed to differentiate further.

Matrigel® Coated Culture Substrates:

Wells were coated with Matrigel® according to manufacturers directions.Briefly, either regular Matrigel® or growth factor reduced Matrigel®(Collaborative Biosciences) was thawed at 4° C. for at least 3 h. It wasdiluted 1:10 or 1:20 in cold KO DMEM for hES cell cultures or 1:30 forhepatocyte cultures. Using pre-cooled plates and pipette tips 0.75-1 mLof Matrigel solution was added to each well (9.6 cm²). The plate wasincubated at room temperature for one h or at 4° C. overnight, and thenwashed once with cold KO DMEM before adding cells.

Immunocytochemistry:

Cells growing on chamber slides were fixed in 3.5% paraformaldehyde for5 min at room temperature, and then for 20 minutes in methanol at −20°C. The fixed cells were rinsed with PBS twice and blocked for 1 hour in10% goat serum in PBS. They were then incubated in primary antibodydiluted in 10% goat serum and PBS for 2 h. Antibody to albulmin, alphafetoprotein (AFP) (Sigma) and α1-antitrypsin (OEB Biosciences Inc.) werediluted at 1:500, cytokeratin, 8, 18 and 19, desmin, (Neomarkers),vimentin (Dako) and SMA (Sigma) were diluted at 1:200. Cells were thenwashed 3 times with PBS and incubated in secondary antibody, which wasFITC-conjugated anti-mouse IgG diluted 1:100 and Hoechst HH33258 (Sigma)at 1:1000 in 5% goat serum in PBS, and incubated for 1 h. The stainedcells were then washed 3 times in PBS, and mounted in Vectashield™(Vector Labs). Images were taken at 10× and 40× using a Nikon Labophot™equipped with epifluorescence and a spot CCD camera.

Glycogen Staining:

Periodic Acid Schiff's stain (PAS) was obtained from American MasterTech Scientific Inc. Cells were grown on chamber slides and fixed inacetone:methanol 1:1 at −20° C. for 20 min. The fixed cells were rinsedin tap water followed by distilled water. The cells were then incubatedin 0.5% periodic acid solution for 4 min at room temperature, and rinsedwith distilled water. They were then incubated with Schiff's solutionfor 10 min at room temperature and rinsed with tap water several times.The cells were then incubated in Fast Green stain for 2 minutes, rinsedtwice with 100% alcohol, and mounted in DPX mounting media. Images weretaken at 10× and 40× using Nikon Labophot™ equipped with epifluorescenceand a spot CCD camera.

BrdU Staining:

Cells were grown on chamber slides in the indicated growth medium, andlabeled with 10 μM BrdU for 24 h. Cells were then fixed with 3:1methanol: acetic acid for 30 minutes, and air-dried overnight in thedark. The fixed cells were rinsed once in PBS, and denatured in 0.07 NNaOH for 2 min followed by quick rinses in PBS pH 8.5 and pH7.4 severaltimes. They were then blocked using 1.5% goat serum (Vector Labs) for 15min, and incubated with anti BrdU antibody (Sigma) diluted 1:500 in 1.5%goat serum and 0.05% Tween™ 20 for 2 h. The samples were washed thricein PBS, and then incubated with secondary antibody, which wasbiotinylated goat anti-mouse immunoglobulin (Vector Labs), at 10 μg/mLdiluted in 1.5% horse serum for 30 min. The sample was washed againthrice in PBS, and then incubated with the staining conjugate, Texas Redlabeled streptavidin (Vector Labs) at 30 μg/mL diluted in 10 mM HEPESbuffer and 0.15M NaCl pH 8.5, for 20 min in the dark. Hoechst HO33258stain (bisbenzimide, Sigma Cat. No. B2883) was mixed into thestreptavidin solution at 2.5 uM final concentration to stain all thenuclei. The stained cells were washed again 3× in PBS, and mounted inVectashield™ (Vector Labs). Images were taken at 10× and 40× using NikonLabophot™ equipped with epifluorescence and a spot CCD camera.

Reverse-transcriptase PCR Amplification:

RT-PCR analysis of expression at the transcription level was conductedas follows: RNA was extracted from the cells using RNAeasy Kit™ (Qiagen)as per manufacturer's instructions. The final product was then digestedwith DNase to get rid of contaminating genomic DNA. The RNA wasincubated in RNA guard (Pharmacia Upjohn) and DNAse I (Pharmacia Upjohn)in buffer containing 10 mM Tris pH 7.5, 10 mM MgCl₂, and 5 mM DDT at 37°C. for 30-45 minutes. To remove protein from the sample, phenolchloroform extraction was performed and the RNA precipitated with 3 Msodium acetate and 100% cold ethanol. The RNA was washed with 70%ethanol, and the pellet was air-dried and resuspended in DEPC-treatedwater. For the reverse transcriptase (RT) reaction, 500 ng of total RNAwas combined with a final concentration of 1× First Strand Buffer(Gibco), 20 mM DDT and 25 μg/mL random hexamers (Pharmacia Upjohn). TheRNA was denatured for 10 min at 70° C., followed by annealing at roomtemperature for 10 min. dNTPs were added at a final concentration of 1mM along with 0.5 μL of Superscript II RT (Gibco), incubated at 42° C.for 50 minutes, and then heat-inactivated at 80° C. for 10 min. Sampleswere then stored at −20° C. till they were processed for PCR analysis.Standard polymerase chain reaction (PCR) was performed using primersspecific for the markers of interest in the following reaction mixture:cDNA 1.0 μL, 10×PCR buffer (Gibco) 2.5 μL, 10×MgCl₂ 2.5 μL, 2.5 mM dNTP3.0 μL, 5 μM 3′-primer 1.0 μL, 5 μm 5′-primer, 1.0 μL, Taq 0.4 μL,DEPC-water 13.6 μL. Selected markers and reaction conditions are shownin Table 3.

TABLE 3 Reaction Conditions for Expression Analysis by RT-PCR ExpectedMcCl₂ Annealing Marker size (mM) temp PCR Cycle α-fetoprotein 157 1.7559° C. (94° C. 30 sec; 59° C. 30 sec; 72° C. 30 sec) × 30 albumin 2331.5 57° C. (94° C. 30 sec; 57° C. 30 sec; 72° C. 30 sec) × 35α₁-antitrypsin 213 1.5 67° C. (94° C. 30 sec; 57° C. 30 sec; 72° C. 30sec) × 35 HNF1a 150 1.5 62° C. (94° C. 3 min) × 1; (94° C. 30 sec; 62°C. 30 sec; 72° C. 30 sec) × 35; (72° C. 10 min) × 1 HNF3β 170 1.5 62° C.(94° C. 3 min) × 1; (94° C. 30 sec; 62° C. 30 sec; 72° C. 30 sec) × 35;(72° C. 10 min) × 1 HNF4a 497 1.5 61 ° C. (94° C. 3 min) × 1; (94° C. 30sec; 61 ° C. 30 sec; 72° C. 30 sec) × 35; (72° C. 10 min) × 1 ASGR 2261.5 60° C. (94° C. 3 min) × 1; (94° C. 30 sec; 60° C. 30 sec; 72° C. 30sec) × 35; (72° C. 10 min) × 1 GATA4 256 1.25 62-70° C. (94° C. 30 sec;70° C. 30 sec) × 35 C/EBPaα 396 1.5 61° C. (94° C. 3 min) × 1; (94° C.30 sec; 61° C. 30 sec; 72° C. 30 sec) × 35; (72° C. 10 min) × 1 C/EBPβ213 1.5 61° C. (94° C. 3 min) × 1; (94° C. 30 sec; 61° C. 30 sec; 72° C.30 sec) × 35; (72° C. 10 min) × 1 β-actin (control) 285 1.5-2.5 55-61 °C. any cycle above

Example 1 Differentiation of Human Embryonic Stem Cells Using n-Butyrate

Embryoid bodies (EB) were prepared as described in the precedingsection. After 5 days in suspension culture, they were harvested andplated on Growth Factor Reduced Matrigel® coated plates and in chamberslides (Nunc). One of the following three conditions was used inparallel:

medium containing 20% fetal bovine serum (FBS);

medium containing 20% FBS and 5 mM sodium butyrate (Sigma);

medium containing 20% FBS, 0.5% DMSO (ATCC), 4 μm dexamethazone (Sigma),150 ng/ml insulin, 10 ng/ml EGF, 600 nM glucagon (Sigma).

In each case, the medium was exchanged every day, and cells were fixedfor immunocytochemistry on day 4 after plating.

One day after plating, the EBs plated in 20% FBS alone looked healthy,almost all of them adhered to the plate and appeared to beproliferating. After several days, the cells in FBS alone survived well,and differentiated to form a very heterogeneous population. In contrast,1 day after plating the cultures containing sodium butyrate had a largeproportion of apparently dead cells, and only some patches comprising afairly homogenous population of cells survived. The morphology of thesecells was similar to that of primary hepatocytes, in that the cells werelarge and became multinucleated after a few days. These cultures werecompared with cultures of primary human hepatocytes (obtained from Dr.Stephen Strom, University of Pittsburgh), and with HepG2 cells (apermanent human hepatocyte cell line derived from a hepatoblastoma,similar to what is reported in U.S. Pat. No. 5,290,684). In thecondition with 0.5% DMSO and growth factors (no. 3), the cells lookedhealthy and the cultures contained a remarkably heterogeneous populationof cells.

FIG. 1 shows the morphology of embryoid body cells replated and culturedfor a further 2 days (4×, 10×, 20×). The right side shows cellsdifferentiated by culturing 2 days in the hepatocyte differentiationagent n-butyrate. A round colony forms at the site where an embryoidbody is plated; the white patch in the middle is a small region of deadcells. The other cells in the field show remarkably homogenousmorphology. The left side shows cells cultured in serum-containingmedium alone. The embryoid body disperses over a wide area, and formsheterogeneous patches of cells that show the morphology of manydifferent cell types.

Four days after plating, cells growing on chamber slides were fixed forimmunocytochemistry, using antibodies against different liver specificmarkers. Results are shown in Table 4. Cultures treated with sodiumbutyrate did not express AFP, but about 30% of the cells expressedantibody-detectable levels of albumin.

TABLE 4 Immunocytochemistry of Cultured Cells primary Embryonic StemCells human specificity of cultured 4 days with hepato- primary antibodyNa butyrate FBS alone FBS + DMSO cytes (none) − − − − non-specific IgG1− − − − AFP − + + − Albumin 30% +ve* − − 100% +ve α₁-antitrypsin >60%+ve + + >80% +ve CK18 100% +ve + + 100% +ve CK8  100% +ve + + 100% +veCK19 100% +ve + + 100% +ve Desmin −  5% +ve 5% +ve (n.d.) Vimentin 100%+ve + + 100% +ve SMA <1% +ve 20% +ve 5% +ve (n.d.) *- results are givenin terms of percentage of cells showing positive staining (n.d.) = notdetermined in this experiment

Example 2 Markers Expressed by Differentiated Cells

hES cell derived embryoid bodies were harvested after 4 or 5 days insuspension, and plated on Matrigel® coated 6-well plates (for RNAextraction) and chamber slides (for immunocytochemistry) in mediumcontaining 20% FBS and 5 mM sodium n-butyrate. The medium was changeddaily or every other day. There was a lot of cell death on day 1followed by less cell death on the subsequent days.

FIG. 2 shows the morphology of the differentiated cells after 6 days ofculture with n-butyrate. Six different fields are shown from the sameculture (10× in the top row, 20× in the other rows). The cells areremarkably uniform, showing a large polygonal surface and binucleatedcenter characteristic of mature hepatocytes.

On the sixth day after plating in the differentiation agent, the cellswere analyzed for expression of markers by RT-PCR andimmunocytochemistry, following the procedures outlined earlier. Glycogencontent in these cells was determined using periodic acid Schiff stain.The number of cells in S phase of cell cycle was determined byincubating the cells with 10 μm BrdU on day 5 after plating, andsubsequently staining with anti-BrdU antibody 24 hours later.

FIG. 3 shows the results of immunohistochemical staining for certaincell specific markers. FIG. 3A (40×) shows the results for primary adulthuman hepatocytes obtained from the University of Pittsburgh—antibodystaining on the right side, Hoechst HH33258 bisbenzimide staining of thesame field for cell nuclei on the left side. FIG. 3B (20×) shows theresults for hES cells cultured 6 days with n-butyrate. Both sets ofcells show staining in a high proportion of cells for albumin,α₁-antitrypsin, and CD18, three markers characteristic of cells of thehepatocyte lineage, and negative for α-fetoprotein which is a marker forearly progenitor cells.

FIG. 4 shows the glycogen staining pattern of cells cultured 6 days inn-butyrate (10× and 40×). The cells were stained with Periodic AcidSchiff's stain for glycogen (pink, dark color) and with Fast Green stainto outline the cell cytoplasm (background green, light color). About 60%of the butyrate treated cells show evidence of glycogen storage (toprow), compared with 80% in fetal hepatocytes (middle row, positivecontrol) and virtually none in the human fibroblast cell line designatedBJ fibroblast (bottom row, negative control).

A summary of the phenotype analysis is provided in Table 5. Albuminexpression was found in 55% of the cells. AFP was completely absent.Glycogen was being stored in at least 60% of the cells. 16% of the cellslabeled with BrdU, indicating that a significant portion of the cellswere proliferating at the time of analysis.

TABLE 5 Phenotype of Differentiated Cells Primary Antibody Specificity %positive cells (none)  0 non-specific IgG1  0 α-fetoprotein  0 Albumin 55% α₁-antitrypsin  90% CK18 100% CK8  100% CK19 100% Desmin  0Glycogen staining  60% BrdU staining 16%

RT-PCR analysis was also performed after six days of culture withn-butyrate to look at the expression pattern of various genes normallyexpressed in hepatocytes. These data were compared with the expressionpattern of the same genes in adult hepatocytes, fetal hepatocytes, HepG2cells (a hepatocarcinoma line) and a non hepatocyte RPE (Retinal pigmentepithelial) cell line. Results are shown in Table 6.

TABLE 6 RTPCR analysis of Gene Expression Embryoid HepG2 primary primaryEmbryoid Embryoid Body hepato- human fetal Body cells Body cells cellsRPE cyte hepato- hepato- cultured in cultured cultured epithelial cellline cytes cytes hES cells FBS with DMSO with cell line (positive(positive (positive (undiffer- (cell and growth sodium (negativecontrol) control) control) entiated) mixture) factors n-butyratecontrol) β-actin + + + + + + + + α-fetoprotein + + + + + + + −albumin + + + − + + + − α₁-antitrypsin + + + − + + + + HNF1a + + + − + +− − HNF3b + + + − + + − − HNF4a + + + − − − − − ASG receptor + + +− + + + − GATA-4 + + + + + + + − C/EBPα + + + − + + + − C/EBPβ + + +− + + + −

The effect of sodium butyrate was compared with other potentialhepatocyte differentiation agents in a similar protocol. Embryoid bodieswere cultured in suspension for 4 days, and then replated on platescoated with collagenase I. The cells were then cultured in the presenceof each compound for 6 days. Results are shown in Table 7.

TABLE 7 Hepatocyte Differentiation Agents Induction of hepatocytephenotype NaCl − n-Butyric Acid + Sodium n-butyrate + α-hydroxybutyricacid − β-hydroxybutyric acid − Propionic acid ± Valeric acid −Isovaleric acid ± Caproic acid − Isobutyric acid ± Trichostatin A + +Causes hepatocyte differentiation and selective elimination of othercell types − No inductive effect ± Mild inductive effect may allowgrowth or survival of other cell types

At a concentration of 5 mM, sodium chloride had no effect, while butyricacid and sodium butyrate were equally effective—indicating that thedifferentiation is not simply due to a change in ion concentration. Thereader will appreciate that butyric acid and [sodium] butyrate areconjugate forms of the same substance that are within the bufferingcapacity of culture media. Accordingly, the terms are interchangeable inthis disclosure unless explicitly required otherwise.

For comparative purposes, a variety of structural analogs of butyratewere tested at 5 mM. The analogs propionic acid, isovaleric acid, andisobutyric acid were effective in causing hepatocyte differentiation,but were deemed less preferable under these conditions becauseenrichment for cells bearing the hepatocyte phenotype was less robust.

Trichostatin A, which is another inhibitor for histone deacetylase, wasfound to be toxic to cells in the range of 2.5-100 μm, and ineffectiveat 10-50 nM. At 75-100 nM, Trichostatin A appeared to both inducehepatocyte differentiation and select against survival of other celltypes. The phenotype of hepatocyte lineage cells made using 5 mM sodiumn-butyrate and 100 nM Trichostatin A is shown in Table 8.

TABLE 8 Phenotype of Differentiated Cells primary hES cells hES cellshuman Primary Antibody differentiated using differentiated using hepato-Specificity Sodium Butyrate Trichostatin A cytes (none)  0%  0%non-specific IgG1  0%  0% α-fetoprotein  0%  0% albumin  62%  41% >80%α₁-antitrypsin  90%  81%  90% CK18 100% >70% 100% CK19 100% >90% 100%Glycogen staining >60% >50% >80%

Example 3 Augmentation of the Differentiating Effect of n-Butyrate withHepatocyte Maturation Factors

The effect of various possible hepatocyte maturation factors was testedin cells differentiated using n-butyrate. hES were cultured for 4 daysin 5 mM sodium n-butyrate, and then switched to a different medium. Thefollowing alternatives were tested:

1. “HCM” medium from Clonetics

2. 10% fetal bovine serum (FBS) supplemented with insulin, epidermalgrowth factor (EGF), dexamethazone, and glucagon;

3. 10% calf serum (CS) supplemented with insulin, EGF, dexamethazone,and glucagon;

4. 20% FBS supplemented with insulin, EGF, dexamethazone, and glucagon

The cells were maintained under these conditions for 4 days. Cellssurvived under all conditions, but appeared best in 10% FBS with growthfactors (Groups 2 and 4). These cells were trypsinized and replated infresh Matrigel® coated plates. Other growth factors are tested in asimilar protocol, or in combination, to determine their effects onhepatocyte maturation and cell phenotype.

Example 4 Telomerization of hES-derived Hepatocytes

Several days after differentiation of hES with sodium butyrate, cellsare transduced with a retrovirus encoding the human homolog oftelomerase reverse transcriptase (hTERT). The vector comprises an hTERTencoding sequence from a plasmid designated pGRN145, into the EcoR1 siteof the commercially available pBABE puromycin construct. The hTERTencoding sequence is placed under control of the retrovirus LTRpromoter. Control and hTERT PBABE retroviral supernatants are preparedusing the PA317 packaging cell line, and combined with 4 μg/mLpolybrene.

Cultures of differentiated hES cells are prepared, and the medium isreplaced with a medium containing retrovirus supernatant for 8-16 hours.The medium is replaced again with normal growth medium, and the cellsare allowed to recover for 1-2 days. Cells are then selected using0.5-2.5 μg/mL puromycin. The cells are evaluated morphologically, forgrowth rate, and for expression patterns using immunocytochemistry andRT-PCR. Telomerase activity is evaluated using the TRAP assay.

Example 5 Differentiation of Human Embryonic Stem Cells in Feeder-freeCulture

Undifferentiated hES colonies were passaged continuously in feeder-freeconditions as follows. Cultures were incubated in 1 mg/mL collagenasefor about 5 minutes at 37° C. The cells were then harvested by scrapingthe cells off the surface and dissociating them into small clumps. Cellswere split at a 1:3 or 1:6 ratio, ˜55,000 cells/mL (17,000 cells/cm²).The day after replating, colonies of undifferentiated cells could againbe identified. Single cells in between the colonies were differentiated.Over the next few days, the undifferentiated cells were seen toproliferate and the colonies became large and compact. Thedifferentiated cells in between the colonies also became more compact.The cells became confluent after 4-7 days of being fed daily withconditioned media. When the cells reached confluence, they were splitonce more.

For this example, the H9 hES cells (p30+5) were maintained infeeder-free conditions for 30 days (5 passages) before differentiation.The undifferentiated cells were maintained on laminin and fed withMEF-conditioned medium, as described elsewhere in this disclosure. Toinduce differentiation, the conditioned medium was replaced with SRmedium (without supplemental bFGF) containing 5 mM sodium butyrate.

After one day in these conditions, small patches of cells could be seenthat had a hepatocyte-like morphology. In addition, a large number ofcells died and appeared to be adhered to the bottom of the dish. In thecontrol cultures that received SR media without butyrate, considerablediversity of differentiation (cells with different morphologies) wasobserved. About six days after treatment, the culture that had receivedsodium butyrate contained many patches of hepatocyte-like cells, but afew cells with other morphologies were identified. Many dead cells stilladhered to the dish. Cultures that did not receive butyrate appearedvery differentiated, with a diversity of phenotypes.

In subsequent experiments, hES cells maintained in feeder-freeconditions are exposed to sodium butyrate at the time of passaging. ThehES cell line designated H9 and maintained for 48 days (8 passages) onMatrigel® is harvested at confluence using collagenase, and reseeded onMatrigel®. The cells are passaged into SR media containing 5 mM sodiumbutyrate, and assessed for hepatocyte-like morphology and geneexpression at various times of culture, in comparison with cellscultured without butyrate.

Example 6 Effect of Butyrate in Combination with DMSO

In this experiment, the effect of the hepatocyte differentiation agentbutyrate was determined in the presence of hepatocyte maturation factorDMSO.

Human ES cell derived embryoid bodies were harvested after 4 days insuspension and plated in the following four conditions.

Gelatin coated plates in the presence of 5 mM Na butyrate

Gelatin coated plates in the presence of 5 mM Na butyrate and 1% DMSO

Matrigel® coated plates in the presence of 5 mM Na butyrate.

Matrigel® coated plates in the presence of 5 mM Na butyrate and 1% DMSO

Media were changed every other day and cells were analyzed on day 7 forimmunocytochemistry and RT-PCR. Cells in all these conditions lookedmorphologically alike, comprising colonies of cells with uniformmorphology. There were fewer colonies of cells in the set where butyrateand DMSO were both present, compared with the ones cultured in butyratealone. The two sets that were plated on gelatin had even less cells.

Immunostaining showed a similar marker phenotype in all the conditions.Percentage of cells in the culture staining for each of the markerstested is shown in Table 9.

TABLE 9 Phenotype of Differentiated Cells Group 1 Group 2 Group 3 Group4 Gelatin Gelatin Matrigel ® Matrigel ® Butyrate Butyrate + DMSOButyrate Butyrate + DMSO No Primary antibody  0  0  0  0 IgG1  0  0  0 0 α-fetoprotein  0  0  0  0 Albumin 56% 75% 50%  63%α₁-antitrypsin >90% >90% >90% >90% CK18 100% 100% 100% 100% CK19 100%100% 100% 100% Glycogen >60% >60% >60% >60%

Example 7 Direct Differentiation of hES to Hepatocyte-like Cells withoutForming Embryoid Bodies

The undifferentiated hES cells were maintained in feeder-free conditions(on Matrigel® in MEF-CM). The strategy was to initiate a globaldifferentiation process by adding the hepatocyte maturation factors DMSOor retanoic acid (RA) to a subconfluent culture. The cells are theninduced to form hepatocyte-like cells by the addition of Na-butyrate.

The hES cells were maintained in undifferentiated culture conditions for2-3 days after splitting. At this time, the cells were 50-60% confluentand the medium was exchanged with unconditioned SR medium containing 1%DMSO. The cultures were fed daily with SR medium for 4 days and thenexchanged into unconditioned SR medium containing 2.5% Na-butyrate. Thecultures were fed daily with this medium for 6 days; at which time onehalf of the cultures were evaluated by immunocytochemistry. The otherhalf of the cultures were harvested with trypsin and replated ontocollagen, to further promote enrichment for hepatocyte lineage cells.Immunocytochemistry was then performed on the following day.

As shown in Table 10, the cells which underwent the final re-plating had˜5-fold higher albumin expression, similar α₁-antitrypsin expression and2-fold less cytokeratin expression than the cells not re-plated. Thesecondary plating for the cells is believed to enrich for thehepatocyte-like cells.

TABLE 10 Phenotype of Differentiated Cells No trypsinizationTrypsinization Antibody Specificity % positive % positive (no primaryantibody)  0  0 (IgG1 control)  0  0 albumin  11%  63%α₁-antitrypsin >80% >80% α-fetoprotein  0  0 Cytokeratin 8  >80 %  45%Cytokeratin 18 >80 %  30% Cytokeratin 19 >80 %  30% glycogen  0 >50%

Example 8 Comparison of Different Matrices for HepatocyteDifferentiation from hES Cells

EBs were generated from hES cells in feeder-free conditions. After 4days in suspension, the EBs were plated in 20% FBS medium supplementedwith 5 mM Na-butyrate or 5 mM Na-butyrate and 1% DMSO. The EBs wereplated on the following matrices:

1. collagen I (0.03 mg/mL coated overnight at 37° C.)

2. growth factor reduced Matrigel® (1:10, coated for 1 h at room temp)

3. gelatin (1% coated for 2 h at 37° C.)

After 6 days in Na-butyrate the cells were evaluated morphologically andusing immunocytochemistry for hepatocyte markers. In all conditions,homogeneous patches of hepatocyte-like cells were observed. However, thenumber of cell clusters was greatly reduced in the cultures with gelatincoating compared with other conditions. As shown in Table 11, thepercentage of cells with albumin, cytokeratin, and α₁-antitrypsinimmunoreactivity was similar in all conditions. Glycogen storage wasalso similar in all conditions. These data indicate that all thesubstrates tested promote hepatocyte differentiation, but Matrigel® andcollagen I coating support survival better than gelatin.

TABLE 11 Phenotype of Differentiated Cells Matrigel ® Gelatin CollagenButyrate + Butyrate + Butyrate + Antibody Specificity Butyrate DMSOButyrate DMSO Butyrate DMSO (no primary) 0  0  0  0  0  0  (IgG1control) 0  0  0  0  0  0  α-fetoprotein 0  0  0  0  0  0  albumin 56%75% 50% 63% 79% 75% α₁-antitrypsin >90%  >90%  >90%  >90%  >90%  >90% Cytokeratin 18 100%  100%  100%  100%  100%  100%  Cytokeratin 19 100% 100%  100%  100%  100%  100%  Glycogen >60%  >60%  >60%  >60%  >60% >60%

Example 9 Further Optimization of Conditions for Direct Differentiation

hES cells undergo the Direct Differentiation protocol detailed earlier,making the adjustments to culture conditions shown in Table 12.Hepatocyte Culture Medium is purchased from Clonetics; Strom's Medium isprepared as described in Runge et al., Biochem. Biophys. Res. Commun.265:376, 1999. The cell populations obtained are assessed byimmunocytochemistry and enzyme activity.

TABLE 12 Direct Differentiation Protocols Further differentiationUndifferentiated cells Pre-differentiation Hepatocyte induction (Groups1-3 only; (until confluent) (4 days) (6 days) 4 days) Feeder-freeconditions 20% SR medium + 20% SR medium + HCM + 30 ng/mL hEGF + 1% DMSO1% DMSO+ 10 ng/mL TGF-α + 2.5 mM butyrate 30 ng/mL HGF + 1% DMS0 + 2.5mM butyrate Feeder-free conditions 20% SR medium + 20% SR medium + 20%SR medium + 1% DMSO 1% DMSO+ 30 ng/mL hEGF + 2.5 mM butyrate 10 ng/mLTGF-α + 30 ng/mL HGF + 1% DMSO + 2.5 mM butyrate Feeder-free conditions20% SR medium + 20% SR medium + Strom' medium + 1% DMSO 1% DMSO + 30ng/mL hEGF + 2.5 mM butyrate 10 ng/mL TGF-α + 30 ng/mL HGF + 1% DMSO+2.5 mM butyrate Feeder-free conditions 20% SR medium + HCM + 30 ng/mLhEGF + 1% DMSO 10 ng/mL TGF-a + 30 ng/mL HGF + 1% DMSO+ 2.5 mM butyrateFeeder-free conditions 20% SR medium + 20% SR medium + 1% DMSO 30 ng/mLhEGF + 10 ng/mL TGF-α + 30 ng/mL HGF + 1% DMSO+ 2.5 mM butyrateFeeder-free conditions 20% SR medium + Strom's medium + 1% DMSO 30 ng/mLhEGF + 10 ng/mL TGF-α + 30 ng/mL HGF + 1% DMSO+ 2.5 mM butyrateFeeder-free conditions HCM + 30 ng/mL hEGF + HCM + 30 ng/mL hEGF + 10ng/mL TGF-α + 10 ng/mL TGF-α + 30 ng/mL HGF + 30 ng/mL HGF + 1% DMSO 1%DMSO + 2.5 mM butyrate Feeder-free conditions 20% SR medium + 20% SRmedium + 30 ng/mL hEGF + 30 ng/mL hEGF + 10 ng/mL TGF-α + 10 ng/mLTGF-α + 30 ng/mL HGF + 30 ng/mL HGF + 1% DMSO 1% DMSO + 2.5 mM butyrateFeeder-free conditions Strom's medium + Strom's medium + 30 ng/mL hEGF +30 ng/mL hEGF + 10 ng/mL TGF-α + 10 ng/mL TGF-α + 30 ng/mL HGF + 30ng/mL HGF + 1% DMSO 1% DMSO + 2.5 mM butyrate

Other additives tested in the subsequent (4-day) maturation step includefactors such as FGF-4, and oncostatin M in the presence ofdexamethazone.

FIG. 5 shows the effect of HCM on maturation of hES-derived cells. Leftcolumn: 10× magnification; Right column: 40× magnification. By 4 days inthe presence of butyrate, more than 80% of cells in the culture arelarge in diameter, containing large nuclei and granular cytoplasm (RowA). After 5 days in SR medium, the cells were switched to HCM. Two dayslater, many cells are multinucleated, and have a large polygonal shape(Row B). By 4 days in HCM, multinucleated polygonal cells are common,and have a darker cytosol (Row C), by which criteria they resemblefreshly isolated human adult hepatocytes (Row D) or fetal hepatocytes(Row E).

Example 10 Metabolic Enzyme Activity

hES-derived hepatocyte lineage cells generated by the directdifferentiation protocol were tested for cytochrome P450 activity.

After completion of the differentiation protocol, cells were culturedfor 24-48 hours with or without 5 μm methylchloranthrene, an inducer forthe cytochrome P-450 enzymes 1A1 and 1A2 (CYP1A1/2). Enzyme activity wasmeasured as the rate of de-ethylation of ethoxyresorufin (EROD). Thesubstrate was added to the medium at a concentration of 5 μM, andfluorescence of the culture supernatant was measured after 2 hours in afluorimetric microplate reader at 355 nm excitation and 581 nm emission.The amount of resorufin formed was determined using a standard curvemeasured for purified resorufin, and expressed as picomoles resorufinformed per min per mg protein.

FIG. 6 shows the results. CYP1A1/2 activity was detected in the threehepatocyte lineage cell lines tested—two derived from the H1 ES cellline, and one derived from the H9 ES cell line. The level of activitywas inducible by methylchloranthrene (MC), and exceeded the levelobserved in two preparations of freshly isolated human adult hepatocytes(HH). The level of activity in undifferentiated H1 and H9 cells (and inthe BJ human embryonic fibroblast cell line) was negligible.

It will be recognized that the compositions and procedures described inthis disclosure can effectively be modified by those skilled in the artwithout departing from the spirit of the invention embodied in theclaims that follow.

What is claimed as the invention is:
 1. A method of screening a compoundfor its effect on hepatocytes or a hepatocyte activity, comprising: a)combining the compound with a cell population obtained bydifferentiating primate pluripotent stem (pPS) cells, wherein at least˜60% of cells in the population have at least three of the followingcharacteristics: antibody-detectable expression of α₁-antitrypsin (AAT);antibody-detectable expression of albumin; absence ofantibody-detectable expression of α-fetoprotein; RT-PCR detectableexpression of asialoglycoprotein receptor (ASGR); ability to storeglycogen; cytochrome p450 activity; glucose-6-phosphatase activity; orthe morphological features of hepatocytes; b) determining any change tocells in the population or their activity that results from beingcombined with the compound; and c) correlating the change with theeffect of the compound on hepatocytes or a hepatocyte activity.
 2. Amethod of screening a compound for its effect on hepatocytes or ahepatocyte activity, comprising: a) combining the compound with a cellpopulation obtained by differentiating primate pluripotent stem (pPS)cells in a medium containing butyrate or an inhibitor of histonedeacetylase; b) determining any change to cells in the population ortheir activity that results from being combined with the compound; andc) correlating the change with the effect of the compound on hepatocytesor a hepatocyte activity.
 3. A method of screening a compound for itseffect on hepatocytes or a hepatocyte activity, comprising: a) combiningthe compound with a cell population containing cells that have the samegenome as an established human embryonic stem (hES) cell line, whereinat least 60% of cells in the population have at least three of thefollowing characteristics: antibody-detectable expression ofα₁-antitrypsin (AAT); antibody-detectable expression of albumin; absenceof antibody-detectable expression of α-fetoprotein; RT-PCR detectableexpression of asialoglycoprotein receptor (ASGR); ability to storeglycogen; cytochrome p450 activity; glucose-6-phosphatase activity; orthe morphological features of hepatocytes; b) determining any change tocells in the population or their activity that results from beingcombined with the compound; and c) correlating the change with theeffect of the compound on hepatocytes or a hepatocyte activity.
 4. Themethod of claim 1, comprising determining whether the compound is toxicto cells in the population.
 5. The method of claim 1, comprisingdetermining whether the compound affects ability of cells in thepopulation to proliferate or be maintained in culture.
 6. The method ofclaim 1, comprising determining whether the compound changes enzymeactivity or secretion.
 7. The method of claim 6, comprising determiningwhether the compound changes activity of a hepatocyte Phase Imetabolizing enzyme.
 8. The method of claim 6, comprising determiningwhether the compound changes activity of a hepatocyte Phase IImetabolizing enzyme.
 9. The method of claim 6, comprising determiningwhether the compound changes cytochrome p450 expression or activity. 10.The method of claim 9, comprising determining whether the compoundchanges CYP3A3-5 activity, CYP2D activity, or CYP2C9 activity.
 11. Themethod of claim 9, comprising determining whether the compound affectsCYP1A1 or CYP1A2 activity.
 12. The method of claim 6, comprisingdetermining whether the compound affects the activity of7-ethoxycoumarin O-de-ethylase, aloxyresorufin O-de-alkylase, coumarin7-hydroxylase, p-nitrophenol hydroxylase, testosterone hydroxylation,UDP-glucuronyltransferase, glutathione S-transferase, γ-glutamyltranpeptidase, or glucose-6-phosphatase.
 13. The method of claim 1,comprising determining whether the compound affects the synthesis of aplasma protein.
 14. The method of claim 13, wherein the plasma proteinis albumin, transferrin, α₁-antitrypsin (AAT), or α-fetoprotein.
 15. Themethod of claim 1, comprising determining whether the compound affectsgluconeogenesis, ureagenesis, bilirubin conjugation, or bile acidconjugation.
 16. The method of claim 1, comprising determining whetherthe compound affects synthesis or secretion of cholesterol orlipoprotein, the level of glutathione, nucleoside phosphate metabolism,intracellular K²⁺ or Ca⁺ concentration, the release of nuclear matrixproteins or oligonucleosomes, induction of apoptosis, or glycogenstorage.
 17. The method of claim 1, wherein the pPS cells are humanembryonic stem cells.
 18. The method of claim 1, wherein cells in thepopulation have been genetically altered.
 19. The method of claim 18,wherein cells in the population have been genetically altered to expresstelomerase reverse transcriptase.
 20. The method of claim 1, wherein atleast ˜60% of the cells in the population have at least five of saidcharacteristics.
 21. The method of claim 1, wherein at least ˜80% of thecells in the population have at least seven of said characteristics. 22.The method of claim 1, wherein the level of cytochrome p450 enzyme1A1/1A2 activity in the cell population is at least as high as inprimary human adult hepatocytes.
 23. The method of claim 1, wherein thecell population has been obtained by culturing the pPS cells in a growthenvironment that comprises a hepatocyte differentiating agent.
 24. Themethod of claim 23, wherein the hepatocyte differentiating agent isn-butyrate or an inhibitor of histone deacetylase.
 25. The method ofclaim 1, wherein the cell population has been obtained by culturingprogeny of the pPS cells in a medium containing one or more hepatocytematuration factors.
 26. The method of claim 25, wherein at least onehepatocyte maturation factor is either: an organic solvent selected fromthe group consisting of dimethyl sulfoxide (DMSO), dimethylacetamide(DMA); hexmethylene bisacetamide, and other polymethylene bisacetamides;or a cytokine or hormone selected from the group consisting ofglucocorticoids, epidermal growth factor (EGF), insulin, TGF-α, TGF-β,fibroblast growth factor (FGF), heparin, and hepatocyte growth factor(HGF), IL-1, IL-6, IGF-I, IGF-II, and HBGF-1.
 27. The method of claim 1,comprising providing the cell population with which the compound issubsequently combined.
 28. The method of claim 1, comprisingdifferentiating an hES cell line to obtain the cell population withwhich the compound is subsequently combined.